首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 31 毫秒
1.
  相似文献   

2.
  相似文献   

3.
The various types of cells that comprise the tumor mass all carry molecular markers that are not expressed or are expressed at much lower levels in normal cells. These differentially expressed molecules can be used as docking sites to concentrate drug conjugates and nanoparticles at tumors. Specific markers in tumor vessels are particularly well suited for targeting because molecules at the surface of blood vessels are readily accessible to circulating compounds. The increased concentration of a drug in the site of disease made possible by targeted delivery can be used to increase efficacy, reduce side effects, or achieve some of both. We review the recent advances in this delivery approach with a focus on the use of molecular markers of tumor vasculature as the primary target and nanoparticles as the delivery vehicle.

Introduction

The concept of targeted drug delivery is attractive because it recapitulates some of the advantages of topical application of drugs: high local concentration and low systemic exposure. In practice, this approach has met with some success but has not provided the hoped-for “silver bullets.” However, recent developments in the field have rekindled interest in the targeting approach. We call this mode of drug delivery “synaphic” targeting; it is also referred to as pathotropic or active targeting. Cancer stands out as a disease most likely to benefit from targeted drug delivery. Tumor cells express many molecules on their surface that distinguish them from normal cells. Traditionally, such molecules were detected with antibodies, but screening of peptide and aptamer libraries has greatly expanded the number of tools available for selective binding to tumor cells (for reviews see Ruoslahti, 2002; Peer et al., 2007). Leukemia and lymphoma treatments with antibodies conjugated to a radioisotope have been in clinical use for several years (Sharkey and Goldenberg, 2005). However, this approach has not been as successful with solid tumors. The apparent reason is the difficulty in delivering drugs into these tumors; drugs only penetrate a few cell diameters into the extravascular tumor tissue from blood vessels (Hambley and Hait, 2009). This low penetration appears to arise from two main factors: first, tumor vessels are poorly perfused with blood and are dysfunctional, which limits the delivery of blood-borne compounds to tumors (Jain, 1999). Second, tumors have a high interstitial pressure thought to result from dysfunctional lymphatics, which causes tissue fluid to flow out of the tumor, working against diffusion of drugs from the blood vessels into the tumor (Jain, 1999; Heldin et al., 2004). The leakiness of tumor vessels partially makes up for the poor penetration (the so-called enhanced permeability and retention [EPR] effect), but EPR is not very effective, and its size dependency and variability from tumor to tumor limit its usefulness (Maeda et al., 2000; Iyer et al., 2006; Sugahara et al., 2009). Interstitial fibrosis can further retard the diffusion of compounds through tumors (Olive et al., 2009). Targeting treatments to selective markers in tumor vessels does not suffer from some of these drawbacks of targeting tumor cells; in particular, no tissue penetration is required for the compound to reach its target. The luminal side of tumor vessels is fully accessible to compounds circulating in the blood, and the vessels can serve as a gateway to the tumor interior for compounds concentrated in the vessels. Using a targeting probe with tumor-penetrating properties and a receptor that is shared between tumor vessels and tumor cells provides additional advantages (Fig. 1). Thus, we have chosen to focus this review on targeting approaches that make use of specific markers in tumor vessels. We will also discuss solutions to the poor penetration of compounds into tumor tissue and the roles that nanoparticles can play in targeted therapies.Open in a separate windowFigure 1.Synaphic targeting of tumors. The targeted receptors can be on tumor cells, tumor vessels, or shared by both. (A) Probes that recognize solely tumor cells provide little improvement of tumor accumulation over a nontargeted probe. (B) Probes that recognize tumor vessels accumulate in the tumor, but entry into tumor tissue relies on passive mechanisms. (C) Probes that recognize both the vessels and tumor cells combine the (limited) efficiency of the two targeting mechanisms. (D) Tumor-penetrating targeting probes (so far only peptides with such characteristics are known) provide a particularly potent targeting system.

Molecular signatures in tumor vessels

Distinct features of tumor vessels.

Tumor blood vessels are distinct from normal vessels. In addition to being tortuous, uneven in diameter, and leaky, tumor vessels express various cell surface and extracellular matrix proteins that normal vessels do not express or do so at much lower levels than tumor vessels (for review see Ruoslahti, 2002). The expression of many of these proteins in tumor vessels is associated with angiogenesis, and they are often functionally important in that process (Hanahan and Folkman, 1996; Alitalo and Carmeliet, 2002). Tumors also contain lymphatic vessels, and many tumors produce growth factors that stimulate lymphangiogenesis (Karpanen and Alitalo, 2008). Lymphatics are not necessary for tumor growth but are important conduits of metastasis. Like tumor blood vessels, tumor lymphatics can also express specific molecular markers.

Screening for markers in tumor vasculature.

Screening of phage-displayed peptide libraries, particularly when performed in vivo, has provided a very useful discovery tool for vascular markers in tumor vessels and elsewhere (Pasqualini and Ruoslahti, 1996). A major advantage of the in vivo phage screening is that it is unbiased in revealing what works in vivo. Other unbiased methods, such as antibody-based screens (Jacobson et al., 1996), cloning strategies (Carson-Walter et al., 2001), and in vivo biotinylation (Borgia et al., 2010), have also been used successfully in analyzing tumor vasculature. Phage screening has uncovered a large number of tumor-homing peptides that have been used to identify the corresponding binding protein (receptor). An early study on tumor-homing peptides (Arap et al., 1998) validated the method by producing tumor-homing peptides with RGD (arginine/glycine/aspartice acid) and NGR (asparagine/glycine/arginine) motifs, which had been previously identified in screens for integrin-binding peptides performed in vitro. F3 is an example of a novel tumor-homing peptide identified by in vivo phage screening (Porkka et al., 2002). F3 binds to nucleolin, which is ubiquitous as an intracellular protein but is expressed at the cell surface of endothelial cells and tumor cells in vivo (Christian et al., 2003). In vitro, all cells seem to be positive for cell surface nucleolin (Borer et al., 1989; Bonnet et al., 1996) presumably because cultured cells resemble cells that have been activated in vivo. Cell surface nucleolin is an angiogenesis marker that is both a suitable target for drug delivery (Christian et al., 2001; Reddy et al., 2006; Henke et al., 2008; Drecoll et al., 2009) and involved in the angiogenesis process (Fogal et al., 2009). The in vivo screening of phage libraries has also produced several potent tumor-homing peptides, the target molecules of which remain to be identified (Hoffman et al., 2003; Joyce et al., 2003; Järvinen and Ruoslahti, 2007; Chang et al., 2009).The expression of intracellular proteins such as nucleolin at the cell surface of tumor cells and tumor endothelial cells appears to be a general principle. Phage display is particularly well suited for the discovery of such markers because the method inherently relies on binding to accessible targets on the cell surface rather than overall expression levels. In vivo cell surface labeling followed by monoclonal antibody production and proteomics analyses is another way of interrogating the cell surface. In addition to aforementioned nucleolin, the cytoplasmic proteins annexin1 (Oh et al., 2004) and plectin-1 (Kelly et al., 2008) have been found to be present at the cell surface of endothelial cells in tumors but not in normal tissues. Another example is p32 protein (gC1q receptor, hyaluronic acid–binding protein). This protein is primarily a mitochondrial protein, but it is also expressed at the cell surface of lymphatic, myeloid, and cancer cells in tumors but not in normal tissues (Fogal et al., 2008). This protein is the receptor for the tumor-homing peptide LyP-1, originally discovered using in vivo phage display (Laakkonen et al., 2002).

Adhesion receptors as angiogenesis markers.

Some of the molecular markers in tumor vasculature have been found by studying the expression of known cell surface receptors in tumor vessels. A prime example is the overexpression of αvβ3 and αvβ5 integrins in angiogenic vessels (Brooks et al., 1994; Erdreich-Epstein et al., 2000; Desgrosellier and Cheresh, 2010). These integrins are prime targets for synaphic drug delivery. Vascular markers expressed on the surface of the endothelium, such as the integrins, are most readily available for the binding of blood-borne compounds. However, the ECM also contains distinct markers that can be used in tumor targeting. An alternatively spliced form of fibronectin containing an additional type III domain, ED-B, is selectively expressed in tumor (and other) angiogenic vessels (Nilsson et al., 2001). Antibodies to ED-B have been used to construct immunotoxins and other compounds for tumor targeting. Proteolytically processed type IV collagen is another matrix component that can be detected with antibodies or peptides (Roth et al., 2006; Mueller et al., 2009). The support cells (mural cells) in the vascular wall also contain markers that are specific for tumor vessels and that can be potentially useful in tumor targeting. NG2, a membrane-spanning chondroitin sulfate proteoglycan, is a cell surface marker of pericytes (and smooth muscle cells) in angiogenic vessels not expressed in the pericytes of normal vessels (Stallcup and Huang, 2008). One of the PDFG receptors is another marker that is expressed at high levels in pericytes (Song et al., 2005).

Fibrin–fibronectin complexes in tumors.

Peptides that specifically bind to fibrin–fibronectin complexes or other proteins associated with these complexes also home to tumors. The walls of tumor vessels and the interstitial spaces in tumors contain products of blood clotting, presumably as a result of plasma protein seepage from leaky tumor vessels. Leaked fibrinogen is converted to a fibrin meshwork by tissue-procoagulant proteins such as tissue factor (Dvorak et al., 1985; Abe et al., 1999; Pilch et al., 2006). Other plasma proteins, plasma fibronectin in particular, become covalently linked or otherwise bound to the fibrin meshwork. These fibrin–fibronectin complexes in the walls of tumor vessels and in the tumor interstitial stroma can be accessed with peptides derived from phage screening, such as the nine–amino acid cyclic peptide CLT-1 (Pilch et al., 2006; Ye et al., 2008) and the pentapeptide CREKA (Simberg et al., 2007). Fibrin-binding peptides isolated for the purpose of targeting blood clots in cardiovascular disease would presumably behave similarly if tested for tumor homing. The CREKA peptide has been used to confer a new function to nanoparticles: self-amplification of tumor homing (see Amplified tumor homing; Simberg et al., 2007).

Tumor endothelial markers (TEMs).

Surveying mRNA expression by the serial analysis of gene expression technique has revealed a large number of striking differences between endothelial cells isolated from human colon cancers and those from adjacent normal tissue (Carson-Walter et al., 2001). Among these TEMs are collagens, some of which are expressed at strikingly high levels in tumor endothelial cells, at least at the mRNA level. The high collagen expression may relate to the extensive fibrosis found in many tumors and recently shown to contribute to the poor penetration of drugs into tumors (Olive et al., 2009). Perhaps the most interesting among the TEMs is TEM 8, which is one of the two receptors for the anthrax toxin (Nanda et al., 2004). An effort is under way to develop anthrax toxin variants that bind only to TEM 8 and that could be used to target tumor vasculature for destruction.
ReceptorReferences
RGD-directed integrins (αvβ3 and αvβ5)Ruoslahti, 2002; Desgrosellier and Cheresh, 2010
Aminopeptidase NPasqualini et al., 2000
TEMsCarson-Walter et al., 2001
EndosialinChristian et al., 2001
Cell surface nucleolinChristian et al., 2003
Cell surface annexin-1Oh et al., 2004
Cell surface p32/gC1q receptorFogal et al., 2008
Cell surface plectin-1Kelly et al., 2008
Fibronectin ED-BNilsson et al., 2001
Fibrin–fibronectin complexesPilch et al., 2006; Simberg et al., 2007
Interleukin-11 receptor αLewis et al., 2009
Protease-cleaved collagen IVXu et al., 2001; Mueller et al., 2009
Open in a separate window

Stage-specific markers.

The molecular angiogenesis signatures vary depending on the state of tumor development. Initiation of angiogenesis (the angiogenic switch) occurs already in premalignant lesions (Hanahan and Folkman, 1996). Peptide probes that distinguish between the blood vessels of premalignant and fully malignant lesions of some de novo cancers in mice have been reported (Hoffman et al., 2003; Joyce et al., 2003). The vascular molecules recognized by these peptides remain to be identified. It may also be possible to develop targeting probes that distinguish between physiological and tumor angiogenesis (Seaman et al., 2007).

Nontumor angiogenesis.

A significant issue in the use of angiogenesis-detecting probes in cancer diagnosis or therapy is that angiogenesis also occurs in regenerating tissues and in inflammation. This poses a potential problem for tumor targeting, as angiogenesis associated with tissue repair in conditions coexisting with cancer, such as myocardial infarction or stroke, could be inadvertently targeted for destruction. This circumstance emphasizes the need to discover vascular markers with more focused target recognition properties. Probes that recognize tumor type–specific markers would fall into this category, as they obviously could not be targeting all forms of angiogenesis. The idea that this level of specificity can be achieved with tumor vessels has been demonstrated with tumor type–specific peptides (Hoffman et al., 2003; Joyce et al., 2003; Laakkonen et al., 2004). Peptides like this should make diagnostic and therapeutic applications possible that are more selective than angiogenesis-based targeting. The use of peptides (or other types of probes) with this kind of focused specificity would likely require first diagnostically assessing the tumor of each individual patient for the selective expression of the appropriate receptor. Such personalized medicine seems certain to become increasingly prevalent.

Functional role of tumor vessel markers.

The αv integrins play an important role in angiogenesis, although the details of their involvement in this process remain to be fully elucidated (Desgrosellier and Cheresh, 2010). Peptides containing an NGR sequence motif had previously been shown to bind weakly to the RGD-binding site of integrins (Koivunen et al., 1994), but this motif was later identified as the binding motif in tumor-homing peptides that were more potent than could be expected on the basis of the weak integrin binding (Arap et al., 1998). It was subsequently shown that the NGR peptides recognize aminopeptidase N (Pasqualini et al., 2000) and potentially, after a chemical alteration, αv integrins (Curnis et al., 2008). Like nucleolin, aminopeptidase N is functionally important in the angiogenesis process (Pasqualini et al., 2000; Rangel et al., 2007). These findings serve as a paradigm to illustrate a discovery process in which a new homing peptide is discovered in phage screening, the receptor for the peptide is identified by biochemical methods such as affinity chromatography, and subsequent studies reveal a role for the receptor in the biology of tumor vessels. Once the receptor is identified, an effective therapy may be engineered. Both F3 and the NGR motif peptides have been used to target drugs to tumors (Curnis et al., 2004; Reddy et al., 2006; Henke et al., 2008), and aptamers that bind nucleolin are being pursued in phase I clinical trials (Laber, D., V.R. Sharma, D.A. Laber, V.R. Sharma, L. Bhupalam, B. Taft, F.J. Hendler, and K.M. Barnhart. 2005. American Society of Clinical Oncology Annual Meeting Proceedings. Abstr. 3064).

Delivery of therapeutic agents to vascular targets

Targeting integrins.

The αvβ3 and αvβ5 integrins are highly expressed in tumor endothelium, and their level of expression may be highest in the vessels of the most malignant tumors (Erdreich-Epstein et al., 2000). Enhanced drug delivery with vascular homing peptides has been accomplished using a cyclic peptide containing the integrin-binding RGD motif (CRGDC) to deliver doxorubicin to tumors (Arap et al., 1998).Remarkable success in targeting the cytokine TNF into tumors has been reported with RGD and NGR peptides; the targeted cytokine was effective in doses as much as 1,000-fold lower than the usual dose and effectively mitigating side effects as a result of the high toxicity of this cytokine (Curnis et al., 2004). These same peptides have also been used to deliver tissue factor to induce blood clotting specifically in tumor blood vessels, with resulting occlusion of the vessels and tumor necrosis (Bieker et al., 2009). A targeted TNF is currently in clinical trials (Paoloni et al. 2009; Gregorc et al., 2010).Conjugates of an antibacterial peptide, which destroys mitochondria in mammalian cells causing apoptosis, with either the RGD or NGR peptide also inhibited tumor growth in mice, whereas either peptide alone was inactive (Ellerby et al., 1999). Moreover, targeting the same proapoptotic peptide to the blood vessels of the normal prostate caused partial destruction of the prostate and delayed the development of cancers in transgenic prostate cancer mice (Arap et al., 2002). The potential of synaphic targeting is very well illustrated; the combination of homing peptides with nonselectively toxic compounds, such as proapoptotic peptides and TNF, can profoundly alter the in vivo activity of the toxins. RGD peptides and antibodies to αvβ3 integrin have also been successfully used in targeted delivery of diagnostic probes to tumors (Sipkins et al., 1998; Stollman et al., 2009; Sugahara et al., 2009), and imaging probes based on this approach are in clinical trials. Drug-loaded nanoparticles have also been targeted with RGD peptides to suppress tumor growth or metastasis (Hood et al., 2002; Murphy et al., 2008; Sugahara et al., 2009). Finally, RGD and other tumor-homing peptides have been used to alter the host range of viral gene therapy vectors (Wickham, 2000; Haviv et al., 2002). Several homing peptides that bind to receptors other than integrins have also been successfully used in preclinical studies to target gene therapy vectors, drugs, and biologicals into tumors (Müller et al., 2003; Hamzah et al., 2008; Chang et al., 2009; Karmali et al., 2009).

Tumor-penetrating peptides.

A major problem with many of the currently used tumor-targeting probes is that a reagent directed to tumor cells will be impeded by the poor permeability of tumors to blood-borne compounds. This problem is particularly prominent with solid tumors, which have a high interstitial pressure, presumably because their blood vessels tend to be leaky and their lymphatic vessels poorly functional (Jain, 1999). Drugs generally do not penetrate further than three to five cell diameters from blood vessels, which leaves more distantly located tumor cells without any drug or exposes them to low drug concentrations that are likely to facilitate the development of resistance (Hambley and Hait, 2009). Despite these limitations, a homing peptide that binds to the Her2 receptor (Gee et al., 2008) has been used to deliver compounds to tumors that overexpress this receptor. Folic acid is another probe commonly used to target the folate receptor, which is overexpressed by tumor cells in many tumors (for review see Salazar and Ratnam, 2007). Experimental and theoretical results indicate that this increase in efficacy is not dominated by changes in overall drug uptake by the tumor (i.e., increased volumetric concentration) but rather changes in cellular internalization of the drug or how long it is retained in the tumor (Bartlett et al., 2007). Thus, there is little or no specific accumulation of probes targeted solely to tumor cells (Fig. 1 A). Results with nanoparticles targeted to tumor cells should be interpreted with particular care. Nanoparticles are small (from a few to 200 nm in diameter) particles that can serve as drug carriers and contrast agents (for imaging) in medicine. Although small in comparison with cells, nanoparticles are much larger than molecules and are less likely to penetrate the vascular wall and gain access to tumor cells than small molecular mass drugs or even antibodies. Tumor blood vessels are more readily available for targeting than the tumor cells and can mediate specific targeting (Fig. 1 B). It is not clear to what extent the Her2 or folate receptors might be expressed on tumor endothelial cells, where they would contribute to the uptake of drugs by the tumor. Some other receptors used in synaphic targeting are expressed both in the tumor vessels and on tumor cells. Examples include αv integrins and nucleolin. These dual targeters are more effective than probes that recognize only the vessels or the tumor cells (Fig. 1 C). However, strategies to increase overall tumor accumulation of drugs and nanoparticles are still needed. Targeting with tumor-penetrating peptides, particularly when the peptide binds both to the tumor endothelium and the tumor cells, provides such a strategy (Fig. 1 D).The laboratory of E. Ruoslahti has recently discovered a tissue–cell penetration system that makes it possible to derive peptides that not only home to a specific target tissue but also penetrate into that tissue. The peptides contain a tissue penetration motif, R/KXXR/K, which has to be exposed at the C terminus of a peptide (or protein) to be active (the C-end rule [CendR]; Teesalu et al., 2009). A tumor-homing CendR peptide contains both a tumor-specific homing sequence and a cryptic (not C terminal) CendR sequence. The homing sequence takes the peptide to the vascular endothelium in the target tissue, where the peptide is proteolytically processed by an endogenous protease such that the CendR motif becomes C terminal and active. The activated CendR motif then binds to a different receptor (neuropilin-1), which mediates extravasation, tissue penetration, and cell entry of the C-terminally truncated peptide and any payload attached to it. An RGD containing a CendR motif, iRGD, exemplifies the capabilities of these peptides. The iRGD peptide penetrates into tumor tissue and is capable of carrying 10 times more drug cargo into a tumor than a conventional RGD peptide (Sugahara et al., 2009).Several aforementioned homing peptides may be tumor-penetrating peptides similar to iRGD. F3 (Porkka et al., 2002), LyP-1 (Laakkonen et al., 2002), and CRGRRST (Joyce et al., 2003) each contain a potential CendR sequence. Moreover, F3 and LyP-1 have been shown to cause extravasation of their cargo, which can be as large as a nanoparticle, with subsequent uptake into tumor endothelial cells and tumor cells (Porkka et al., 2002; Laakkonen et al., 2004; Karmali et al., 2009). Coating of abraxane, which is a nanoparticle drug composed of paclitaxel and albumin, with the LyP-1 or iRGD peptide made the drug capable of penetrating into tumor tissue, resulting in several-fold higher activity than that of the original drug (Karmali et al., 2009; Sugahara et al., 2009). The tissue-penetrating properties of these peptides and their internalization into cells makes them particularly efficient in achieving a high concentration of the peptide and any payload attached to it in tumor tissue. Unlike the cell-penetrating peptides related to the human immunodeficiency virus Tat protein, which do not display any cell type specificity (Gump and Dowdy, 2007), the CendR tumor-homing peptides are tumor specific. Jiang et al. (2004) have described a peptide design in which a negatively charged sequence tethered to a cationic cell-penetrating peptide blocks the cell-penetrating activity until a tumor protease cleaves the tether. The authors achieved a threefold increase in tumor homing. The greater tumor-homing selectivity of peptides derived from in vivo phage display such as iRGD (12-fold) is likely because of the presence of a homing sequence (RGD in iRGD) in these peptides (Sugahara et al., 2009).

Limitations of synaphic targeting

Receptor capacity.

An important factor to consider in synaphic tumor targeting is the capacity of the receptors that are targeted by the probe. The number of cell surface receptors and their availability determine how many molecules of a targeting compound can be specifically bound at the tumor site. Under ideal conditions (infinite binding affinity), the amount of compound that can be bound by the tumor equals the number of available receptors (assuming a 1:1 binding ratio and negligible turnover). For example, assuming a tumor cell volume of one nanoliter and the presence of 100,000 receptors per cell, there would maximally only be a total of 0.166 nmol of receptor per gram of tumor. The binding of the targeting ligand for the receptor is likely to have a high nanomolar to low micromolar disassociation constant, which means that more of the targeting compound has to be administered than can be accommodated by the receptors to drive the interaction toward binding and receptor saturation. Moreover, only a fraction of the receptors is likely to be available to bind a ligand introduced into the blood stream. Any excess of the targeting compound is going to be handled by the body like any other nontargeted compound. If the amount of free targeting compound substantially exceeds the receptor-bound amount, the effect of specific targeting will be drowned out by nonspecific background. This circumstance, illustrated in Fig. 2, is underappreciated in the field, despite repeated demonstrations that targeted compounds are more differentially active when administered at low doses. One potential solution to this problem is to use higher affinity ligands for the targeting, but this strong binding can lead to reduced tumor penetration through the so-called binding site barrier (van Osdol et al., 1991; Thurber et al., 2008). Other potential solutions include using anticancer agents with higher potency than most current drugs, using nanoparticle delivery vehicles that deliver more drug per receptor occupied than one to one conjugates, or inducing more binding sites in the tumor (see Amplified tumor homing).Open in a separate windowFigure 2.Saturation of receptors affects the specificity of synaphic targeting. Once the receptors of the homing peptide have been saturated, the specificity of the targeting declines (adapted from experimental data in Kranenborg et al., 1998). au, arbitrary units.

Monovalent versus multivalent binding.

Low affinity of a ligand for its receptor can seriously limit the targeting efficiency or even make it unachievable. Making the low affinity ligand multivalent can circumvent this problem. Multiple weak interactions produce strong binding. Many natural processes, such as antibody interactions, rely on this principle. For example, because each of the six binding sites in IgM antibodies is generally of low affinity, IgM antibodies rely on multivalent binding. Cells adhere through multivalent interactions between integrins and adhesion proteins such as fibronectin. Phage display with cells in vitro or tissues in vivo as the target (Hoffman et al., 2003) is a prime example of a system that probes this moderate affinity, multivalent landscape. Thus, phage display complements other target discovery methods such as those based on antibodies (Jacobson et al., 1996; Oh et al., 2004) or cloning methods (Seaman et al., 2007). The enhanced avidity from multivalency is usually the result of an unaffected binding rate (on rate [kon]) but a reduction in off rate (koff) for the multiple interactions. Multivalency is important in nanoparticle-based targeting because nanoparticles generally carry more than one targeting ligand and are therefore capable of multivalent binding. This concept is particularly relevant for peptides, which typically bind to their targets with relatively modest (low micromolar) affinities. Reulen et al. (2009) converted a nonbinding variant of a collagen-binding protein into an active targeting probe by inserting multiple copies of the protein into a micelle, artificially producing a multivalent ensemble. The reported enhancements from multivalency are as large as 108 but are more typically 10–104. Interestingly, as few as four RGD peptides could provide a 25-fold enhancement in binding of a 30-nm particle to endothelial cells, and just three folate groups led to a 2,500-fold enhancement in dendrimer binding to a surface (dendrimers are branched synthetic polymers that can form nanoparticles and present ligands in a multivalent fashion in which the valency can be readily controlled; Montet et al., 2006; Hong et al., 2007). At the same time, multivalent peptide presentation can increase recognition of nanoparticles by the reticuloendothelial system (RES; also known as the mononuclear phagocyte system [MPS]).Multivalency may partially explain the remarkable 1,000-fold increase in the antitumor activity of TNF observed when homing peptides recognizing tumor vessels were added to the protein (Curnis et al., 2004). TNF is a trimeric protein, which would render the homing peptide multivalent. Another factor in the increase of activity may be that the chimeric compound will presumably engage both the homing peptide and TNF receptors and that this may result in synergistic binding. It will be interesting to see whether other antitumor compounds with their own cell surface receptor, such as the Her2 antibody trastuzumab, would also benefit from homing peptide targeting in this manner.

The stability of targeting probes.

Elimination from the circulation and degradation are among the main factors that determine the efficiency of a targeting probe. Short in vivo half-life can be an advantage in imaging because it quickly eliminates the background caused by excess probe. However, in drug targeting, short half-life gives the targeted drug less time to penetrate into the target tissue. Long circulation times are especially important when the target is outside the vasculature, although tumor-penetrating peptides offer a potential solution to this problem (Sugahara et al., 2009). The half-life primarily depends on the rate of elimination into the urine (small molecules) and uptake by the RES in the liver and spleen (particles). Coupling a small molecular mass drug or probe to polyethylene glycol is commonly used to increase molecular mass above the kidney filtration cut-off size of 5 nm (Choi et al., 2007). Polyethylene glycol coating is also a strategy used to minimize elimination of protein therapeutics. Preventing RES uptake is particularly important when nanoparticles are used in drug delivery.RES, which is also known as MPS, resides primarily in the liver, spleen, and lymph nodes. It eliminates foreign materials, particularly particles, including synthetic nanoparticles, from the circulation. Tumor-responsive, cleavable stealth coatings have been used to mitigate this problem (Harris et al., 2008), but the RES/MPS uptake of nanoparticles remains a major problem in the use of nanoparticles in nanomedicine. It limits the amount of drug or probe that can reach the intended target, obscures the imaging of the liver and the organs near it, and is a source of potential liver toxicity. Coating of nanoparticles with plasma proteins that mediate binding to Kupffer cell receptors in the liver is thought to underlie this phenomenon, but the unfortunate fact is that the molecular mechanisms of the uptake of nanoparticles by the RES are not really understood (for review see Moghimi et al., 2001). It has been empirically shown that particle charge (anionic or neutral), size (<100 nm), and ability to prevent complement binding can reduce rates of RES uptake and extend circulation time in mice (for review see Peer et al., 2007). Other results suggest that the RES uptake may have little to do with plasma protein–mediated opsonization (Simberg et al., 2009). The likely explanation for why this has been such an intractable problem is that the Kupffer cell receptors use multiple low affinity interactions to capture nano (and micro)-particles, rendering conventional receptor identification methods impotent in addressing this issue. The current stealth technologies to make nanoparticles unrecognizable by the RES only delay the inevitable uptake by this system. It is of major importance to nanomedicine that efficient ways of prolonging nanoparticle circulation be discovered.

Targeted delivery of nanoparticles

Regardless of the limitations of nanoparticles, nanoparticle technology offers an exciting platform for drug delivery: they can incorporate unique functions that cannot be engineered into simple drugs. Although both drugs and nanoparticles can be targeted to a tumor, nanoparticles can be engineered to perform more complex, cooperative targeting functions. We will next discuss self-amplified homing of nanoparticles and amplification of the targeting by nanoparticle combinations.

Amplified tumor homing.

We have made use of a peptide that binds to fibrin–fibronectin complexes in blood clots to design a nanoparticle that self-amplifies its own homing to tumors. Iron oxide nanoparticles coated with the CREKA peptide bind and accumulate in tumor vessels where they cause additional clotting (Simberg et al., 2007). The approach is similar to clotting induced in tumor vessels by tumor-targeted tissue factor (Huang et al., 1997; Bieker et al., 2009) with the exception that the CREKA system is based on self-amplified nanoparticle homing. The clotting induced by the CREKA-coated iron oxide particles creates more binding sites for the peptide, which causes more clotting and so on. The 20% occlusion of tumor vessels initially obtained greatly improved tumor imaging. Recent modifications in the system have increased the occlusion rate to 60–70%, producing highly significant inhibition of tumor growth (Agemy, L., K.N. Sugahara, V.R. Kotamraju, K. Gujraty, C. Aleman, R. Nussinov, and E. Ruoslahti. 2009. Proceedings of the 100th Annual Meeting of the American Association for Cancer Research. Abstr. 3668). Importantly, although the CREKA nanoparticles are nonspecifically taken up in the RES, no clotting was seen in the vessels of the RES organs (or any other normal organs), indicating that the clotting mechanism active in this self-amplifying targeting system requires the tumor environment. As this system at this point only leverages the inherent properties of the targeted nanoparticles, it could be further engineered to carry a drug.The ability of one structural type to perform multiple medical diagnostic or therapeutic functions is an advantageous characteristic of nanomaterials that cannot be achieved with organic small molecules. However, nanosystems that integrate multiple functions into a single structure can display reduced efficacy of the separate functions because of space and surface chemistry limitations and increased susceptibility to phagocyte uptake. Engineering separate nanomaterials that synergistically cooperate in their functions, such as tumor homing, is a way of dealing with this problem. This approach is particularly advantageous in combination therapies, which are commonly used in cancer treatmentsAs was discussed earlier, it would be advantageous to create more binding sites for targeted delivery in a tumor, particularly if they are within the vascular space. We recently constructed a system that leverages a biological cascade in vivo to increase the available binding sites for targeted delivery. Plasmonic nanomaterials, such as gold nanorods, present exciting opportunities for such targeting combinations. These materials are metallic structures that efficiently convert optical radiation into heat by coupling into one or more plasmon modes (Hirsch et al., 2003; Hu et al., 2006). We have recently shown that photothermal heating mediated by tumor-targeted gold nanorods can increase binding sites for targeted delivery with thermosensitive drug carriers (Fig. 3; von Maltzahn et al., 2009; Park et al., 2010). Other biological cascades, such as the protease activity that activates CendR peptides in tumors (Sugahara et al., 2009), can be exploited. Imaging of tumors provides another good example of where the combined properties of tumor-targeted nanodevices can potentially improve the treatment of cancer patients. A tumor-targeting nanosystem that possesses both superparamagnetic and fluorescent quantum dot domains offers the possibility to provide a low resolution anatomical reference to guide the surgical procedure (by magnetic resonance imaging) and a high resolution mapping that can be visualized during surgery to identify surgical margins (by fluorescence imaging of quantum dots; Wang et al., 2004; Kim et al., 2005; Sathe et al., 2006; Kim and Taton, 2007; Song et al., 2007; Park et al., 2008; Ye et al., 2008). Recently, we have also designed iron oxide nanoparticles (nanoworms; Park et al., 2009b) with improved properties and nontoxic silicon-based quantum dots (Park et al., 2009a) for such purposes.Open in a separate windowFigure 3.Treating tumors with cooperative nanoparticles. This scheme illustrates a method to induce cooperative nanoparticle behavior that results in more effective delivery of treatments to tumors. This example uses a two-component system consisting of gold nanorods and targeted, thermally sensitive liposomes. (A) Passive accumulation of gold nanorods. The circulating nanorods passively accumulate in the tumor as a result of leakiness of the tumor vasculature (the EPR effect). (B) Laser irradiation of nanorods activates tumor cells. The gold nanorods absorb laser energy, heating the surrounding tissue. This localized rise in temperature increases tissue permeability and induces expression of receptor proteins on the surface of the tumor cells. (C) Targeted nanoparticles (liposomes) bind to tumor. Receptor-specific targeting peptides attached onto the secondary nanoparticles allow these particles to bind to the overexpressed receptor proteins on the heat-activated tumor cells. (D) Activation of targeted liposomes releases drug. In this example, thermally responsive liposomes containing a drug payload are heated with a second laser pulse, inducing rupture of the liposome shell and release of its contents.

Conclusion and future prospects

The concept of synaphic targeting with a “magic bullet” to treat cancer has been around for 100 years but has not met the high expectations placed on it. There appear to be several reasons for the modest success of the approach. One is the early focus on targeting the tumor cells, which has been largely stymied by poor penetration of the tumor cell–binding probes into extravascular tumor tissue. Targeting molecular markers that are specific for tumor vasculature does not suffer from this limitation. The realization that the vasculature is more accessible to molecular probes has been a significant advance. There are several compounds that target receptors in tumor vasculature and, in some cases, both tumor vasculature and tumor cells. However, poor tumor penetration still limits the activity of these compounds. The recently described tumor-penetrating peptides may solve the problem with access to the extravascular tumor tissue. The second major limitation of synaphic targeting discussed in this review is the limited capacity of the receptors to which the targeting probes bind, especially when most of the receptors are unavailable for binding because of limited penetration of the probes into tumor tissue. The tumor-penetrating peptides can make receptors available in parts of a tumor not accessible to conventional probes. Finally, tumor-homing nanosystems that amplify tumor homing can also improve the delivery of compounds to tumors, providing imaging and therapeutic options that were previously unavailable.  相似文献   

4.
A critical evaluation of the predicted and X-ray structures of alpha-lactalbumin     
K. Ravi Acharya  David I. Stuart  David C. Phillips  Harold A. Scheraga 《Journal of Protein Chemistry》1990,9(5):549-563
The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine -Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon -Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C of residues 1–95, 1.1 Å).  相似文献   

5.
N2-fixing cyanobacteria: Why they do not become dominant in shallow hypertrophic lakes   总被引：1，自引：0，他引：1  
Wanda Zevenboom 《Aquatic Ecology》1982,16(2-3):289-290
Summary Phytoplankton species shifts and succession phenomenona in lakes of increasing trophic state were considered, using the basic information on the growth kinetics of the species involved. One of the most obvious signs of advanced eutrophication is the dominance of cyanobacteria (blue-green algae). Striking examples are the shallow, hypertrophic Dutch lakes The Veluwerandmeren (e.g., Wolderwijd and Veluwemeer), whereOscillatoria agardhii, a non-N2-fixing cyanobacterium, has become dominant over the green algae, diatoms and N2-fixing cyanobacteria (BERGER, 1975).We have studied the natural population ofO.agardhii during the growing season, by using physiological indicators, and could adduce that the natural population was successively growing under phosphorus, light, or nitrogen limitation (ZEVENBOOM and MUR, 1978a,b; ZEVENBOOMet al., 1982). One might expect that during the period of nitrogen limitation the N2-fixing speciesAphanizomenon flos-aquae would be favoured and would be able to outgrow the nitrogen-limitedO.agardhii. However, in these lakes,A. flos-aquae was present only in few numbers and a succession fromO. agardhii toA. flos-aquae did not occur. Although field observations may give some indication, they cannot give decisive answers to the question which factor is triggering the observed species shifts and species dominance in natural waters. Such answers can only be obtained from growth kinetic and physiological data of the species involved. In our opinion, the most important factor to consider is the availability of light energy, which decreases with increasing eutrophication.The hypothesis was proposed by Mur and coworkers (MURet al., 1978) that in hypertrophic lakes the prevailing light conditions (low light irradiance) are more favourable forO.agardhii, since this species has a much lower requirement of light energy for growth than green algae as a consequence of its lower specific maintenance rate constant, e (VAN LIERE, 1979; GONS, 1977). Competition experiments, performed withO. agardhii andScenedesmus protuberans under lightlimiting conditions, confirmed the hypothesis (MURet al., 1978), Continuous culture experiments withA. flos-aquae showed that also this species had a higher energy requirement thanO. agardhii (ZEVENBOOM, 1980). The differences were not found in the value of e, but in the growth efficiency. The higher energy requirement ofA.flos-aquae was expected, since energy is needed for heterocyst production and N2 fixation. Under light-limiting conditions and nutrient sufficiency (including nitrogen-nitrate) it can thus be expected that the N2-fixer will be outcompeted by the non-N2-fixing cyanobacterium. This was indeed observed (ZEVENBOOM et al., 1981).We further investigated the competitive interactions betweenA.flos-aquae, O. agardhii andS. protuberans under different sets of irradiance values and nitrate concentrations. We used the growth kinetic data of the species involved, which were obtained by means of continuous culture experiments (GONS, 1977; VAN LIERE. 1979; VAN LIERE and MUR, 1979; GONS and MUR, 1980; ZEVENBOOM and MUR, 1980; ZEVENBOOMet al., 1980; ZEVENBOOMet al., 1981). The competing species could be placed along the gradients of light irradiance values and nitrate concentrations, their positions being defined by their energy requirements and half-saturation constants for nitrate-limited growth, respectively. Distinct niches for the three species were found with respect to light and nitrate. Under conditions of low irradiance values and low (realistic) nitrate concentrations, nitrogen-limitedO.agardhii was able to outgrowA. flos-aquae andS. protuberans as a consequence of its low energy requirement and its high affinity for nitrate. The growth rates of the last two species were restricted by the limited availability of light. However, at high irradiance values,O.agardhii was inhibited in its growth rate and therefore failed to outgrow the other two species. The competition was then restricted to nitrogen-limitedS.protuberans and light-limitedA.flos-aquae; the latter could dominate at low nitrate concentrations. The results of competition experiments withO.agardhii andA.flos-aquae under different sets of irradiance values and nitrate concentrations agreed well with the niche-model described above (Zevenboom, unpubl. results).In conclusion, kinetic data of growth, obtained with continuous culture experiments, can provide basic information to explain species shifts and dominance in lakes with increasing eutrophication. Nitrogen-limiting conditions favour N2-fixing cyanobacteria only when sufficient light is available for their growth (in less hypertrophic waters). The trophic state is thus of major importance and decisive with regard to which species will dominate.  相似文献   

6.
The force and effect of cell proliferation     
Pedro Campinho  Carl‐Philipp Heisenberg 《The EMBO journal》2013,32(21):2783-2784
EMBO J 32: 2790–2803 doi:10.1038/emboj.2013.197; published online September102013The spatiotemporal control of cell divisions is a key factor in epithelial morphogenesis and patterning. Mao et al (2013) now describe how differential rates of proliferation within the Drosophila wing disc epithelium give rise to anisotropic tissue tension in peripheral/proximal regions of the disc. Such global tissue tension anisotropy in turn determines the orientation of cell divisions by controlling epithelial cell elongation.Oriented cell divisions play important roles in the establishment of the animal body plan by both influencing tissue morphogenesis and generating cellular diversity. Generally, the direction of the cell division plane is determined by the orientation of the mitotic spindle prior to cytokinesis. The observation that the mitotic spindle in most animal cell types aligns with the cell''s longest axis has led to the formulation of the ‘long-axis-rule'', postulating that cell shape anisotropy is the main determinant of spindle orientation (Minc et al, 2011). However, cell shape anisotropy is unlikely to be the only determinant since many cell types round up during mitosis, thereby losing their shape anisotropy and others do not follow the long-axis-rule at all. In such cases, division orientation is determined by the polarizing activity of biochemical signals originating from the environment (reviewed in Morin and Bellaïche, 2011). In addition, externally applied forces have also been suggested to control division orientation of single cells in culture independently from their effect on cell shape (Fink et al, 2011).Epithelial growth implies that cells divide parallel to the tissue plane with both daughter cells remaining integrated within the tissue. Although it has been recognized that defects in apico-basal polarity lead to spindle misalignment and disruption of epithelial architecture, the molecular mechanisms underlying this regulation are still unknown. Recent work in the Drosophila wing disc epithelium uncovered that the junctional proteins Scribbled and Discs large 1 (Dlg1) are required for proper spindle alignment parallel to the tissue plane (Nakajima et al, 2013). Similarly, in the Drosophila follicular epithelium, spindle orientation is dependent on the lateral localization of Dlg1, independently of its role in apico-basal polarity (Bergstralh et al, 2013). While such mechanisms ensure that cells divide parallel to the epithelial plane, other mechanisms must still be present to determine the orientation of the mitotic spindle within this plane.In the Drosophila wing disc epithelium, symmetric cell divisions preferentially align with the proximal-distal (PD) axis, thus elongating the organ along this axis (Baena-López et al, 2005). This preferential cell division orientation is determined by the Fat-Dachsous pathway, which promotes accumulation of the atypical myosin Dachs at PD cellular junctions. The polarized activity of Dachs in turn drives cell elongation along the PD axis, leading to a preferential orientation of the mitotic spindle along this axis (Mao et al, 2011). In this issue of The EMBO Journal, Mao et al (2013) report that while mitotic cells located in central regions of the wing disc indeed elongate and divide along the PD axis, cells located in the periphery (proximal edge) elongate and divide orthogonally to the PD axis (Figure 1). These results suggested some type of global planar tissue polarization in proximal regions of the wing disc overriding the local effects of Dachs on spindle orientation. By using laser ablation to reveal tissue tension, the authors showed that in peripheral/proximal regions of the wing disc, junctions oriented orthogonal to the PD axis (PD junctions) are under higher tension than junctions oriented along this axis (lateral junctions; Figure 1). This led them to hypothesize that anisotropic tissue tension might control division orientation of proximal wing cells. Through a combination of elegant genetic experiments and theoretical modelling, the authors then demonstrated that this global tension anisotropy in the proximal wing disc arises from higher cell division rates observed in central versus proximal regions of the wing disc. Furthermore, this apparent tension anisotropy causes concentric elongation of proximal wing disc cells orienting their mitotic spindle orthogonal to the PD axis (Figure 1).Open in a separate windowFigure 1Differential rates of cell division between distal (green) and proximal (red) regions of the Drosophila wing disc epithelium (1) give rise to global patterns of tension anisotropy within the tissue (2). This tension anisotropy promotes cell elongation along the main axis of tension, thereby controlling the orientation of cell division via cell shape anisotropies in proximal regions of the wing disc (3); D, distal; P, proximal.Collectively, these results demonstrate that differential proliferation rates within a tissue can generate global tension anisotropies, which promote cell shape changes that again influence cell division orientation. Further dissection of the mechanisms by which tissue tension controls cell division orientation will clarify if anisotropic tension controls division orientation solely through cell elongation, or if additional mechanosensing mechanisms exist that more directly convey tissue tension information to the mitotic spindle. It might also be worth exploring whether cell divisions along the main axis of tension within the wing disc affect global tension anisotropy, and whether the formation of anisotropic tension around areas of cell proliferation affects the rate of cell division therein. Such interplay between tissue tension anisotropy and cell division orientation/rate will likely be critical for maintaining physiological degrees of tissue tension and growth.In general, the work by Mao et al (2013) provides compelling evidence for a functional link between tissue tension and cell division orientation in a physiological relevant context, paving the way for future studies addressing the reciprocal relationship between these two aspects in tissue morphogenesis.  相似文献   

7.
Comment on: “root orientation can affect detection accuracy of ground-penetrating radar”     
Yuan Wu  Li Guo  Wentao Li  Xihong Cui  Jin Chen 《Plant and Soil》2014,380(1-2):441-444

Introduction

In a recent paper, Tanikawa et al. Plant Soil 373:317–327, (2013) reported a considerable impact of root orientation on the accuracy of root detection and root diameter estimation by ground-penetrating radar (GPR).

Methods

In Tanikawa et al. Plant Soil 373:317–327, (2013), buried root samples in a sand box were scanned from multiple cross angles between root orientation and GPR transecting line under controlled conditions. Changes in radar waveform parameter of roots to different cross angles were investigated.

Results

Tanikawa et al. Plant Soil 373:317–327, (2013) clarified that 1) the variation in amplitude area (a signal strength related waveform parameter) to different cross angles fitted a sinusoidal waveform; and 2) the impact of root orientation on root diameter estimation by GPR could be mathematically corrected by applying a grid transect survey. However, we found that the quantitative relationship established in Tanikawa et al. Plant Soil 373:317–327, (2013) between amplitude area and cross angle was incorrect, and the application of a grid transect survey still underestimated root diameter.

Conclusion

The change in amplitude area to cross angle between transecting line and root orientation fits a sinusoidal waveform but different to that reported in Tanikawa et al. Plant Soil 373:317–327, (2013). The polarization of GPR wave may explain such sinusoidal variation in amplitude area to cross angle. The effect of root orientation on GPR-based root diameter estimation remains to be calibrated.  相似文献   

8.
Order of class III genes relative to HLA genes determined by the haplotype method   总被引：1，自引：0，他引：1  
Alan N. Wilton  Brett Charlton 《Immunogenetics》1986,24(2):79-83
The B18 C4A3 C4BQ0 BfF1 DR3 haplotype was found to be ideal for determining the order of C4 and Bf relative to HLA-B and DR by the haplotype method. All the copies of this haplotype are assumed to be derived from a single ancestral haplotype. Sixteen of the twenty-six BfFl-containing haplotypes carried all of the alleles from this ancestral haplotype. Most of the other BfFl-containing haplotypes could be derived from the ancestral haplotype by a single crossover event for one of the two possible gene orders. This suggests that B18 C4A3 C4BQ0 BfFl DR3 is the sole source of the BfFl allele. The uncommon C4 type on B18 C4A3 C4BQ0 BfFl DR3 facilitates recognition of the BfFl-containing products of recombination between Bf and C4. One such recombinant haplotype was found which shows that the orientation of the class III genes is as follows: C4 is closest to HLA-B and Bf is closest to HLA-DR. This gene order is supported by all the earlier unequivocal results obtained using the haplotype method (Olaisen et al. 1983, Marshall et al. 1984a). Combining these results with the information on class III genes obtained from overlapping cosmid clones (Carroll et al. 1984) and earlier mapping studies (Robson and Lamm 1984) shows that HLA-B is telomeric to 21B. C4B, 21A, C4A, Bf and C2 then follow 21B in that order covering 120 kb. HLA-DR is located further toward the centromere.  相似文献   

9.
Removal of Heavy Metals from Sewage Sludge Used as Soil Fertilizer     
MARIAN TUREK  TEOFIL KOROLEWICZ  JERZY CIBA 《Soil & Sediment Contamination》2005,14(2):143-154
This work has examined sewage sludge of the following heavy metal concentrations (mg/kg): Cd-3.43; Co-5.25; Cu-131; Fe-51300; Mn-177; Ni-37.5; Pb-104; Zn-3300. Metals speciation by sequential extraction according to Tessier et al. (1979), and Rudd et al. (1988), and a procedure recommended by European Community Bureau of Reference (BCR) (Ure et al., 1993; Quevauiller et al., 1996; Davidson et al., 1999), as well as analysis of chemical forms of metals, have been carried out. It has been found that only Zn concentration is higher than the value permissible for agricultural sewage sludge application (2500 mg/kg). The results obtained by Tessier et al. (1979) Tessier, A., Campbell, P. C. and Bisson, M. 1979. Sequential extraction procedure for the speciation of particulate trace metals.. Anal. Chem., 51: 844–851. [Crossref], [Web of Science ®] [Google Scholar], and BCR procedures (Ure et al., 1993; Quevauiller et al., 1996; Davidson et al., 1999) appeared to be consistent. A comparison of the sequential analysis and the analysis of chemical forms of metals indicates that the sum of metal concentrations for the exchangeable, carbonate and bound to Fe/Mn oxyhydroxides forms (found by Tessier et al., 1979, and BCR analyses (Ure et al., 1993; Quevauiller et al., 1996; Davidson, et al., 1999)) corresponds to the sum of sulfate, oxide, metallic and siliceous forms. The concentrations of the forms bound to organic matter or sulfides correspond to the sulfide form while the residue corresponds to the ferrate form. Preparative extraction of metals from the sewage sludge using sodium salt of ethylenediaminetetraacetic acid (EDTA-Na), sodium pyrophosphate (V) and ammonia water has also been investigated. As far as the examined leaching agents are concerned, EDTA-Na appeared to be the best. Single leaching with this agent results in the following metal concentrations remaining in the sludge (mg/kg): Cd-1.1; Co-2.1; Cu-105; Fe-17700; Mn-28.3; Ni-12.8; Pb-44; Zn-1200. They meet the requirements of Polish regulations concerning the use of sewage sludge as a soil fertilizer.  相似文献   

10.
102 Genome engineering nucleases derived from GIY-YIG homing endonucleases     
David Edgell  Benjamin Kleinstiver  Jason M. Wolfs  Li Wang  Tomasz Kolaczyk  Brendon McDowell 《Journal of biomolecular structure & dynamics》2013,31(1):64-65
Efficient targeted manipulation of complex genomes requires highly specific endonucleases to generate double-strand breaks at defined locations (Bibikova et al., 2003; Bogdanove and Voytas, 2011). The predominantly engineered nucleases, zinc-finger nucleases (ZFNs), and TAL effector nucleases (TALENs) use the catalytic domain of FokI as the nuclease portion. This domain, however, functions as a dimer to nonspecifically cleave DNA meaning that ZFNs and TALENs must be designed in head-to-head pairs to target a desired sequence. To overcome this limitation and expand the toolbox of genome editing reagents, we used the N-terminal catalytic domain and interdomain linker of the monomeric GIY-YIG homing endonuclease I-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), and I-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al. 2012). We also made I-TevI fusions to LAGLIDADGs homing endonucleases (I-Tev-LHEs). All the three fusions showed activity on model substrates on par with ZFNs and TALENs in yeast-based recombination assays. These proof-of-concept experiments demonstrate that the catalytic domain of GIY-YIG homing endonucleases can be targeted to relevant loci by fusing the domain to characterize DNA-binding platforms. Recent efforts have focused on improving the Tev-TAL platform by (1) understanding the spacing requirements between the nuclease cleavage site and the DNA binding site, (2) probing the DNA binding requirements of the I-TevI linker domain, and (3) demonstrating activity in mammalian systems.  相似文献   

11.
Linear dichroism of single crystals of the reaction center from Rhodobacter sphaeroides wild type strain 2.4.1     
Harry A. Frank  Carol A. Violette  Shahriar S. Taremi  David E. Budi 《Photosynthesis research》1989,21(2):107-116
The linear dichroism of single crystals of the photochemical reaction center from Rhodobacter sphaeroides 2.4.1, expressed as the anisotropy (or polarization) ratio, p = (A – A )/A + A , relative to the long morphological axis of the crystals, has been measured to be –0.12±0.03 for the primary donor Q y and -0.15±0.8 for the carotenoid. These dichroic effects can be predicted using data obtained from magnetophotoselection (Frank et al. 1979, McGann and Frank 1985) and electron spin resonance (ESR)(Frank et al. 1988a, Budil et al. 1988) experiments. Magnetophotoselection data yield the projections of the transition moments onto the primary donor triplet state principal magnetic axis system. The single crystal triplet state ESR experiments provide the Euler matrix for the transformation from the principal magnetic axis system to the crystal unit cell axis system. Thus, the projections of the transition moments (site 1) onto the crystal units cell axes (a, b, c) are determined to be-0.39, 0.90 and 0.18, respectively. The projections of the carotenoid transition moment (site 1) onto the crystal unit cell axes (a, b, c) are determined to be -0.60, 0.02 and 0.80, respectively. This information used in conjunction with the crystalline space group symmetry (P212121) and the morphology of the crystals allows one to predict the observed anisotropy ratios.  相似文献   

12.
Role of 2-ketobutyrate as an alarmone in E. coli K12: inhibition of adenylate cyclase activity mediated by the phosphoenolpyruvate: glycose phosphotransferase transport system   总被引：4，自引：0，他引：4  
Jacques Daniel  Evelyne Joseph  Antoine Danchin 《Molecular & general genetics : MGG》1984,193(3):467-472
Summary 2-ketobutyrate and its analogues were found to inhibit strongly and transiently the rate of -galactosidase synthesis in Escherichia coli K12. This effect was ascribed to a strong and transient inhibition of the adenylate cyclase activity. By using pts mutants, we showed, in agreement with our previous results (Daniel et al. 1983), that the likely target of 2-ketobutyrate and its analogues is the phosphoenolpyruvate: glycose phosphotransferase transport system (PTS). Furthermore, evidence for such a cascade effect caused by 2-ketobutyrate and its analogues allowed us to corroborate our previous proposal (Daniel et al. 1983) that 2-ketobutyrate, a precursor of isoleucine, acts as an E. coli alarmone monitoring the passage from anaerobic to aerobic growth conditions.  相似文献   

13.
Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma     
Lynne H. Thomas  V. Trevor Forsyth  Adriana ?turcová  Craig J. Kennedy  Roland P. May  Clemens M. Altaner  David C. Apperley  Timothy J. Wess  Michael C. Jarvis 《Plant physiology》2013,161(1):465-476
In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.Growth and form in plants are controlled by the precisely oriented expansion of the walls of individual cells. The driving force for cell expansion is osmotic, but the rate and direction of expansion are controlled by the mechanical properties of the cell wall (Szymanski and Cosgrove, 2009). Expanding, primary cell walls are nanocomposite materials in which long microfibrils of cellulose, a few nanometers in diameter, run through a hydrated matrix of xyloglucans, pectins, and other polymers (Knox, 2008; Mohnen, 2008; Szymanski and Cosgrove, 2009; Scheller and Ulvskov, 2010). Native cellulose microfibrils are partially crystalline (Nishiyama, 2009; Fernandes et al., 2011). Formerly, primary wall cellulose was thought to have a unique crystal structure called cellulose IV1 (Dinand et al., 1996), but NMR evidence suggests the presence of forms similar to the better characterized cellulose Iα and Iβ crystalline forms together with large quantities of less ordered cellulose (Wickholm et al., 1998; Sturcová et al., 2004; Wada et al., 2004). Nevertheless, cellulose is much more ordered than any other component of the primary cell wall (Bootten et al., 2004), in keeping with its key role of providing strength and controlling growth.The stiffness of the cell wall is greatest in the direction of the cellulose microfibrils, where growth is directional and the predominant microfibril orientation is usually transverse to the growth direction (Green, 1999; MacKinnon et al., 2006; Szymanski and Cosgrove, 2009). Expansion of the cell wall then requires either widening of the spacing between microfibrils (Marga et al., 2005) or slippage between them (Cosgrove, 2005), or both, and the microfibrils reorient toward the direction of growth (Anderson et al., 2010). Polymer cross bridges between microfibrils (McCann et al., 1990) are thought to resist these deformations of the cell wall nanostructure and, thus, to control the rate of growth. Until recently, most attention was focused on bridging xyloglucans, hydrogen bonded to microfibril surfaces (Scheller and Ulvskov, 2010). However, there is evidence that not all xyloglucans are appropriately positioned (Fujino et al., 2000; Park and Cosgrove, 2012a) and that other bridging polymers may be involved (Zykwinska et al., 2007). It has also been suggested that bundles of aggregated microfibrils, not single microfibrils, might be the key structural units in primary cell walls (Anderson et al., 2010), as in wood (Fahlén and Salmén, 2005; Fernandes et al., 2011). If so, single microfibrils could bridge between microfibril bundles. In summary, the growth of plant cells is not well understood, and we need more information on how cellulose orientation is controlled and on the nature of the bridging polymers, the cellulose surfaces to which these polymers bind, and the cohesion between microfibril surfaces that might mediate aggregation.Cellulose microfibrils are synthesized at the cell surface by large enzyme complexes having hexagonal symmetry, sometimes called “rosettes” (Somerville, 2006). Each complex contains multiple cellulose synthases that differ between primary cell walls and wood, although the appearance of the complexes is similar (Somerville, 2006; Atanassov et al., 2009). The simultaneous synthesis, from the same end, of all the chains in a native cellulose microfibril is why they are parallel (Nishiyama et al., 2002, 2003), in contrast to the entropically favored antiparallel structure found in man-made celluloses like rayon (Langan et al., 2001). The number of chains in a microfibril and the number of cellulose synthases in the synthetic complex are evidently related. It is commonly assumed that the number of chains is divisible by six, matching the hexagonal rosette symmetry, and 36-chain models (Himmel et al., 2007) bounded by the hydrophilic [110] and [1-10] crystal faces, as in algal celluloses (Bergenstråhle et al., 2008), have been widely adopted. The assembly and orientation of cellulose are connected, as several cellulose synthase mutants have phenotypes defective in cellulose orientation and plant form as well as depleted in cellulose content (Paredez et al., 2008). In certain other mutant lines, the crystallinity of the microfibrils appears to be affected (Fujita et al., 2011; Harris et al., 2012; Sánchez-Rodríguez et al., 2012).Therefore, a detailed understanding of the structure of primary wall cellulose microfibrils would help us to understand cellulose synthesis as well as the growth and structural mechanics of living plants (Burgert and Fratzl, 2009). Primary cell walls and their cellulose skeletons also affect food quality characteristics like the crispness of salad vegetables and apples (Malus domestica; Jarvis, 2011). When biofuels are produced from lignocellulosic biomass, lignification leads to recalcitrance (Himmel et al., 2007), but some of the cell types in Miscanthus spp., switchgrass (Panicum virgatum), and arable crop residues have only primary walls with no lignin, and recalcitrance then depends on the nature of the cellulose microfibrils (Beckham et al., 2011).A relatively detailed structure has recently been proposed for the microfibrils of spruce (Picea spp.) wood (Fernandes et al., 2011), which are 3.0 nm in diameter, allowing space for only about 24 cellulose chains. Evidence from x-ray diffraction supported a “rectangular” shape (Matthews et al., 2006) bounded by the [010] and [200] faces. There was considerable disorder increasing toward the surface, and the microfibrils were aggregated into bundles about 15 to 20 nm across, with some, but not all, of the lateral interfaces being resistant to water (Fernandes et al., 2011). Disordered domains are a feature of other strong biological materials such as spider silk (van Beek et al., 2002).Therefore, it is of interest whether any of these features of wood cellulose might also be found in the cellulose microfibrils of primary (growing) cell walls. It would be particularly useful to characterize the disorder known to be present in primary wall microfibrils, that is, to define how cellulose that is not measured as “crystalline” differs from crystalline cellulose. Many of the experiments leading toward a structure for wood cellulose were dependent on exceptionally uniform orientation of the cellulose microfibrils (Sturcová et al., 2004; Fernandes et al., 2011). However, in growing cell walls, the microfibrils are not uniformly oriented. When microfibrils are first laid down at the inner face of the primary cell wall, their orientation is normally transverse to the direction of growth, but as the cell wall expands, the microfibrils reorient so that the orientation distribution, integrated across the thickness of the expanded cell wall, becomes progressively closer to random (Cosgrove, 2005; MacKinnon et al., 2006).This technical problem does not apply to the cell walls of celery (Apium graveolens) collenchyma, which are similar in composition to other primary cell walls but have their microfibrils oriented relatively uniformly along the cell axis (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b). Some structural information on celery collenchyma cellulose has already been derived from spectroscopic and scattering experiments (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b), confirming the disorder expected in a primary wall cellulose. Some of these experiments were analogous to what has been done on spruce cellulose (Fernandes et al., 2011), but insufficient data are available to specify the number of chains in each primary wall microfibril, the nature and location of the disorder, and the presence or absence of direct contact between microfibrils. Here, we report x-ray and neutron scattering and spectroscopic experiments addressing these questions and leading to a proposed structure for primary wall cellulose microfibrils. Characterizing a structure containing so much disorder presented unusual challenges, but disorder appears to be central to the enigmatic capacity of primary wall cellulose to provide high strength and yet to permit and control growth.  相似文献   

14.
Serum-Cholesterin,ABO-Blutgruppen und Hämoglobintyp     
Gebhard Flatz 《Human genetics》1970,10(4):318-328
Zusammenfassung Serum-Cholesterin, ABO-Blutgruppen (N=715), Glucose-6-PhosphatDehydrogenase (G-6-PD, Farbstoff-Reduktionstest, N=611) und der Hämoglobintyp (osmotische Resistenz und Cellulose-Acetat-Elektrophorese, N=469) wurden bei anscheinend gesunden, 20 Jahre alten Männern aus 12 Distrikten der Provinz Chiang Mai in Nordthailand bestimmt. Das Körpergewicht hatte keinen Einfluß auf die Cholesterinkonzentration. Probanden der Blutgruppe A hatten signifikant höhere Cholesterinwerte als die der Gruppen 0 und B. Gruppe B hatte höhere Werte als Gruppe 0, aber die Differenz war nur schwach signifikant. Der mittlere Cholesterinwert der Probanden mit -Thalassaemia minor war signifikant niedriger als der der Gruppen mit normalem Hämoglobin und mit -Thalassaemie oder abnormalem Hämoglobin. Zwischen den drei letzteren Gruppen bestand kein signifikanter Unterschied. Diese Befunde bestätigen für eine tropische Bevölkerung mit an Fetten armer Ernährung die Beziehung zwischen -Thalassämie (Fessas et al., 1963; Mayo et al., 1969, Griechenland) und ABO-Blutgruppen (Mayo et al., 1969; Oliver et al., 1969; Langman et al., 1969; Beckman u. Olivecrona, 1970) einerseits und der Serum-Cholesterin-Konzentration.
Serum-cholesterol, AB0 blood-groups and haemoglobin typeGenetic influences on the serum-cholesterol level
Summary Serum-cholesterol, ABO blood-groups (N=715), glucose-6-phosphate dehydrogenase (G-6-PD, dye decolorization test, N=611) and haemoglobin type (osmotic fragility, cellulose acetate electrophoresis, N=469) were determined in apparently health, 20 years old males from 12 districts of the province of Chiang Mai in northern Thailand. Body weight and G-6-PD deficiency did not seem to influence the serum-cholesterol level. Probands with blood-group A had significantly higher cholesterol concentrations than groups 0 and B. The difference between groups 0 and B, the latter having somewhat higher levels, was only weakly significant. Cholesterol levels were significantly lower in probands with -thalassaemia minor when compared with a normal control group. The difference between the control group and the probands with -thalassaemia and abnormal haemoglobins (mainly HbE trait) was not significant. These findings confirm for a tropical rural population with a diet low in fat the association between -thalassaemia and low cholesterol concentrations previously reported from Greece (Fessas et al., 1963; Mayo et al., 1969) and the association between blood-group A and high cholesterol levels found in several European populations (Mayo et al., 1969; Oliver et al., 1969; Langman et al., 1969; Beckman and Olivecrona, 1970).

Beurlaubt von der Universitäts-Kinderklinik Bonn.

Eingerichtet und unterstützt von der Stiftung Volkswagenwerk.  相似文献   

15.
Cell biology in development: The cell biology of planar cell polarity     
Danelle Devenport 《The Journal of cell biology》2014,207(2):171-179
Planar cell polarity (PCP) refers to the coordinated alignment of cell polarity across the tissue plane. Key to the establishment of PCP is asymmetric partitioning of cortical PCP components and intercellular communication to coordinate polarity between neighboring cells. Recent progress has been made toward understanding how protein transport, endocytosis, and intercellular interactions contribute to asymmetric PCP protein localization. Additionally, the functions of gradients and mechanical forces as global cues that bias PCP orientation are beginning to be elucidated. Together, these findings are shedding light on how global cues integrate with local cell interactions to organize cellular polarity at the tissue level.The collective alignment of cell polarity across the tissue plane is a phenomenon known as planar cell polarity (PCP). Exemplified by the uniform orientation of bristles covering the insect epidermis or of the hairs covering the mammalian body surface (Fig. 1 A), PCP patterns can align over thousands, even billions of cells. This phenomenon is controlled by the so-called PCP pathway, which integrates global directional cues to produce locally polarized cell behaviors. There has been a recent surge in interest in PCP after discoveries that various processes such as vertebrate gastrulation, mammalian ear patterning and hearing, and neural tube closure all require a conserved set of PCP genes (Heisenberg et al., 2000; Tada and Smith, 2000; Wallingford et al., 2000; Kibar et al., 2001; Murdoch et al., 2001; Curtin et al., 2003; Montcouquiol et al., 2003; Copley et al., 2013). Since that time, the PCP pathway has been found to coordinate cell behaviors in numerous diverse settings including polarized ciliary beating in the trachea and brain ventricles (Tissir et al., 2010; Vladar et al., 2012), oriented cell divisions (Gong et al., 2004; Baena-López et al., 2005; Ségalen et al., 2010; Mao et al., 2011), lung branching (Yates et al., 2010), and hair follicle alignment (Guo et al., 2004; Devenport and Fuchs, 2008), to name a few (Fig. 1). Genetic disruptions to PCP cause severe developmental abnormalities in vertebrates, notably neural tube defects, left/right patterning defects, and ciliopathies, which highlights the essential requirement for PCP in development (Kibar et al., 2001; Murdoch et al., 2001; Curtin et al., 2003; Wang et al., 2006a,b; Kim et al., 2010; Song et al., 2010).Open in a separate windowFigure 1.Planar cell polarity and the core PCP components. (A and B) The Drosophila wing blade and mammalian epidermis illustrate the phenomenon of PCP. In both cases, hairs point in a single direction along the tissue axis, where they align locally with neighboring hairs and globally across the tissue. Whereas Drosophila wing hairs are produced by single cells, mammalian hairs emerge from multicellular hair follicles, which orient as a unit. A conserved PCP pathway controls the collective alignment of both types of structures. (C) Core PCP components localize to the plasma membrane and asymmetrically segregate along the epithelial plane as indicated.Like many types of cell polarity, the establishment of PCP involves (1) a global orienting cue, (2) asymmetric segregation of dedicated polarity proteins, and (3) translation of polarity information into polarized outputs. But unlike other types of cell polarity, the PCP mechanisms we currently understand involve coupling between adjacent cells, allowing for the alignment of polarity over many cell distances.First described in insects and then genetically dissected in Drosophila melanogaster, PCP was long confined to the realm of experimental embryology and genetics until the discovery that the protein products of several PCP genes were localized asymmetrically within the cell, thrusting PCP into the domain of cell biology (for review see Strutt and Strutt, 2009). The challenge to understanding PCP on a molecular level is that long-range PCP is, in essence, an in vivo phenomenon that is difficult to recapitulate in a tissue culture dish. However, recent advances in imaging technology combined with increasingly sophisticated genetic tools are helping us to decipher the in vivo cell biology of PCP. In this review, I highlight some of the recent advances made toward understanding the cell biology underlying the establishment of coordinated polarized cell behaviors. For clarity, I limit discussions of PCP phenomena that meet the definition of PCP proposed by Goodrich and Strutt (2011): namely, that “cell–cell communication causes two or more cells to adopt coordinated polarity” in a process that is mechanistically “dependent upon planar polarity proteins.” Other aligned cellular patterns or examples of noncanonical Wnt signaling, sometimes described as “Wnt/PCP” signaling, will not be discussed.

PCP components and molecular asymmetries

Two molecular systems control PCP behavior, the “core” and the “Fat–Dachsous (Ft–Ds)” PCP pathways. A key feature of both is the asymmetric distribution of their constituents (Fig. 2). The core PCP pathway is composed of the multipass transmembrane proteins Frizzled (Fz), Van Gogh (Vang; also known as Strabismus/Stbm), and Flamingo (Fmi; also known as Starry night/Stan), and the cytosolic components Dishevelled (Dsh), Prickle (Pk), and Diego (Dgo). On one edge of the cell reside Fz, Dsh, and Dgo, and on the opposite side lie Vang and Pk (Figs. 1 C and 2 B; Axelrod, 2001; Strutt, 2001; Feiguin et al., 2001; Tree et al., 2002; Bastock et al., 2003). The atypical cadherin, Fmi, resides on both sides, where it forms homodimers between neighboring cells (Usui et al., 1999; Shimada et al., 2001). These molecular asymmetries are observed in sensory hair cells of the vertebrate inner ear (Wang et al., 2005, 2006a,b; Montcouquiol et al., 2006; Deans et al., 2007; Song et al., 2010), the mammalian epidermis (Devenport and Fuchs, 2008; Devenport et al., 2011), brain ventricles (Tissir et al., 2010), and trachea (Vladar et al., 2012). Mutations in core PCP components lead to a loss or randomization of polarity and misalignment of cellular structures along the tissues axis.Open in a separate windowFigure 2.Asymmetric localization of PCP components. (A) PCP asymmetry develops progressively from an initially uniform distribution of core PCP proteins. Fz, Dsh, and Dgo (red) localize to the distal/posterior edge, whereas Vang and Pk (turquoise) localize to the proximal/anterior side. Fmi (dark blue) localizes to both sides, where it forms homodimers between neighboring cells. (B) Feedback interactions between core PCP components. A Fz–Fmi complex interacts preferentially with a Vang–Fmi complex between cells, whereas proximal and distal complexes antagonize one another within the cell. (C) Ds and Fj are expressed in opposing gradients in the Drosophila wing blade. Fj positively modulates Ft activity, leading to a gradient of Ft activity across the wing (not depicted). (D) Graded expression of Ds and Fj leads to asymmetric cellular localization of Ds and Ft, which form heterodimers between adjacent cells. Dachs, a downstream component of the Ft–Ds pathway, also localizes asymmetrically in association with Ds.The Ft–Ds pathway includes the large protocadherins Ft and Ds and the Golgi resident transmembrane kinase, Four-jointed (Fj; for review see Matis and Axelrod, 2013; Thomas and Strutt, 2012). Similar to the core system, Ft–Ds also displays molecular asymmetries in flies. Ds and its ligand Ft accumulate on opposite cell edges, where they form intercellular heterophilic interactions (Fig. 2 D; Matakatsu and Blair 2004; Ambegaonkar et al., 2012; Brittle et al., 2012). Unlike the core components, Ds and Fj are expressed in complementary gradients in the Drosophila eye and developing wing, which contribute to the cellular asymmetries of Ds and Ft (Fig. 2, C and D). Whether Ft–Ds–Fj gradients and asymmetries are conserved in vertebrate systems has yet to be determined.

Segregation of cortical polarity proteins: Shaking hands with the enemy

The asymmetric segregation of Fz–Dsh–Fmi and Vang–Pk–Fmi complexes to opposite sides of the cell relies on their mutual exclusion intracellularly and their preferential binding between neighboring cells (Fig. 2 B; for review see Strutt and Strutt, 2009). There is mutual interdependence among core PCP components for their asymmetric localization. Depletion of any one core PCP component results in a loss of asymmetry of all the others. In addition, PCP asymmetry develops progressively from initially uniform distributions (Fig. 2 A). Thus, PCP asymmetry can be thought of not as a simple hierarchy of interactions, but the result of feedback amplification of an initial directional bias.

Intercellular interactions.

PCP requires cell–cell communication, mediated by the transmembrane components of the core system, where it is thought that Fz–Fmi on one cell interacts with Vang–Fmi on its neighbor. These interactions are best understood in the Drosophila wing blade, where PCP controls the alignment of wing hairs along the proximal–distal axis (Figs. 1 A and 2, A and B). In the wing, Vang–Pk localize to the proximal face of each cell, whereas Fz–Dsh–Dgo localize distally adjacent to the wing hair (Figs. 1 C and 2, A and B; Axelrod, 2001; Strutt, 2001; Tree et al., 2002; Bastock et al., 2003; Das et al., 2004). By generating mutant clones and examining PCP localization at the clone border, the intercellular interactions between neighboring cells can be assessed in vivo. For example, when Fz is lacking within a clone, leaving only Vang–Fmi available at cell junctions, then Fz–Fmi in adjacent wild-type cells is recruited to the clone border (Chen et al., 2008). Vang mutant clones produce a similar effect, but in this case the excess Fz recruits Vang to clone borders (Bastock et al., 2003). What mediates these intercellular asymmetric interactions? One possibility is that Vang and Fz interact directly, and in vitro binding assays between the Fz extracellular domain and Vang suggest that this mechanism is possible (Wu and Mlodzik, 2008). However, mutants of Fz or Vang lacking their extracellular domains can still recruit one another between cells, which suggests that something else must bridge the two proteins (Chen et al., 2008). The seven-pass transmembrane cadherin, Fmi, likely performs this function. Fmi is essential for the junctional recruitment of Fz and Vang, and Fmi homodimers appear to be functionally asymmetric (Chen et al., 2008; Strutt and Strutt, 2008; Struhl et al., 2012). Clonal overexpression of Fmi preferentially recruits Fz to the clone border, even in the absence of Vang, which suggests that excess or unpaired Fmi is in a configuration that has higher affinity for Fmi–Fz than Fmi–Vang (Chen et al., 2008; Strutt and Strutt, 2008). Thus, Fmi may exist in two forms depending on whether it is paired with Fz or Vang, but the molecular basis for this difference is not known (Chen et al., 2008; Strutt and Strutt, 2008; Struhl et al., 2012).

Amplification of asymmetry.

Intercellular Fz–Fmi and Vang–Fmi complexes can form between cells in any orientation, so how do they resolve into discrete and opposed asymmetric domains? One way is through clustering of Fz–Fmi and Vang–Fmi complexes of the same orientation, and the cytoplasmic PCP components are particularly important for this function. As PCP complexes grow increasingly asymmetric, they cluster into discrete puncta that are stably associated with the plasma membrane and are resistant to endocytosis (Strutt et al., 2011). FRAP analysis of Fz-containing puncta demonstrated that they are highly stable compared with diffuse Fz-GFP, and have limited lateral mobility within the membrane. In the absence of Dsh, Pk, or Dgo, the size, intensity, and stability of Fz-containing puncta are diminished (Strutt et al., 2011), whereas overexpression causes Fz accumulation and coalescence into larger puncta (Feiguin et al., 2001; Tree et al., 2002; Bastock et al., 2003). Although the precise mechanisms driving PCP puncta formation are not known, the cytoplasmic components do not affect endocytosis, which suggests that they contribute to puncta formation by clustering intercellular complexes (Strutt et al., 2011). Pk can interact homophilically (Jenny et al., 2003; Ayukawa et al., 2014), which might promote clustering of proximal Vang–Pk–Fmi complexes. It will also be interesting to determine whether the cytoskeleton is directly connected to PCP complexes to minimize lateral mobility within the membrane.A second mechanism contributing to PCP asymmetry is directed transport. Live imaging of fluorescently tagged PCP proteins in pupal wings showed that Fz- and Dsh-containing particles travel across the cell in a proximal-to-distal direction (Shimada et al., 2006; Matis et al., 2014; Olofsson et al., 2014). These particles most likely represent endosomes undergoing transcytosis, as they arise from the proximal cortex and are labeled by the endocytic tracer FM4-64. This mechanism could serve to amplify asymmetry or even provide the initial polarity bias by removing proximal Fz–Dsh–Fmi complexes and transporting them to the distal side. Directed PCP transport is mediated by an array of subapical, noncentrosomal microtubules (MTs) that align along the proximal–distal axis, with the plus ends oriented with a slight distal bias (Hannus et al., 2002; Shimada et al., 2006; Harumoto et al., 2010; Matis et al., 2014; Olofsson et al., 2014). Ft and Ds are required for proximal–distal MT alignment (Harumoto et al., 2010), which suggests that the Ft-Ds system may feed into the core PCP system by orienting cytoskeletal architecture to deliver Fz–Dsh–Fmi complexes to the distal edge of the cell.Directed transport of Vang-containing endosomes has not been reported in flies, but selective trafficking could target Vang to specific membrane domains. In mammalian cells, exit of the Vang homologue Vangl2 from the trans-Golgi network (TGN) requires Arfrp1 (an Arf-like GTPase) and the clathrin adaptor complex AP-1, neither of which are required for the transport of a mammalian Fz homologue Fz6 or Fmi/Celsr1, which suggests that the differential sorting of PCP complexes to opposite sides of the cell could initiate at TGN export (Guo et al., 2013). Whether newly synthesized Vang and Fz proteins are transported to opposing cell surfaces from the TGN has not yet been explored.Microtubule orientation also correlates with PCP asymmetry in mouse trachea epithelial cells, where PCP coordinates the alignment of motile cilia (Vladar et al., 2012). MTs are planar polarized with their plus ends oriented toward the Fz–Dvl domain, and disruption of MTs with nocodazole impairs core PCP localization. Similarly, MTs are needed to establish Pk asymmetry in gastrulating zebrafish embryos (Sepich et al., 2011). However, in the skin epithelium, MTs align perpendicular to the axis of PCP asymmetry (unpublished data). Thus, directed transport along MTs may not be required in all tissue types for the establishment of PCP asymmetry.

Negative regulation.

Repulsive interactions between Vang- and Fz-containing complexes may also contribute to the amplification of asymmetry, and cytoplasmic proteins have been proposed to perform this function. Pk and Dgo both bind to Dsh in vitro, interacting with the same domain on Dsh in a mutually exclusive manner (Jenny et al., 2005). In addition, overexpression of Pk can prevent Dsh translocation to the membrane (Tree et al., 2002; Carreira-Barbosa et al., 2003), which suggests that Pk binding to Dsh could displace it from the proximal side of the cell. On the distal side, Dgo binding to Dsh would prevent association with Pk, thus enhancing Dsh distal localization. This increase in Dsh and Pk asymmetry would then positively feed back by clustering the transmembrane components into stable membrane domains.Modulation of PCP protein levels by ubiquitin-mediated degradation also leads to feedback by restricting the amount of one PCP protein to antagonize another. In flies, regulation of Dsh by a Cullin-3-BTB E3 ubiquitin ligase complex limits its levels at cell junctions (Strutt et al., 2013a). Reduction of Cullin-3 leads to an increase in overall core PCP protein levels, a reduction of asymmetry, and defects in wing hair polarity, which is consistent with Dsh overexpression phenotypes (Strutt et al., 2013a). SkpA, a subunit of the SCF E3 ligase, regulates Pk levels by promoting its degradation in a Vang-dependent manner (Strutt et al., 2013b). In mice, Smurf E3 ligases ubiquitinate Pk and promote its local degradation by binding to phosphorylated Dvl2 (a mammalian homologue of Dsh; Narimatsu et al., 2009). Smurfs are required for Pk localization in the inner ear and floor plate, and their removal leads to defects in convergent extension (CE) and stereocilia alignment (Narimatsu et al., 2009). Thus, targeting Pk for degradation either balances total Pk protein levels or targets a specific pool of Pk for ubiquitination and proteasome degradation.

Tissue-level polarity cues: This way or that?

What provides the tissue-level polarity cue that biases core PCP asymmetry in one direction over another? This is perhaps the most fundamental, yet poorly understood, element of PCP. Current models propose that an upstream, graded cue provides an initial bias in PCP asymmetry by regulating the levels, localization, or activity of one or more of the core proteins. Gradients are attractive candidates for providing global polarity cues, as they can act across many cells and define the tissue boundaries over which polarity must be oriented.

Ft–Ds–Fj.

Unlike the core proteins, Ds and Fj are nonuniformly expressed in the Drosophila eye, wing, and abdominal segments, and as such, the Ft–Ds module has been proposed to provide a global polarity cue (Fig. 2, C and D; for review see Ma et al., 2003; Yang et al., 2002; Thomas and Strutt, 2012; Matis and Axelrod, 2013). Ft and Ds are heterodimeric cadherins, regulated by the Golgi kinase Fj (Ishikawa et al., 2008; Brittle et al., 2010; Simon et al., 2010). The complementary expression patterns of Ds and Fj are thought to give rise to asymmetric Ft and Ds protein localization, with Ft and Ds localizing to opposite sides of each cell (Fig. 2 D; Ambegaonkar et al., 2012; Bosveld et al., 2012; Brittle et al., 2012). Because Fj positively regulates the activity of Ft, a gradient of Ft activity is expressed across the wing complementary to that of its ligand, Ds (Simon et al., 2010). Mutations in the Ft–Ds system give rise to swirling wing hair patterns, and disrupt the global alignment of core PCP proteins, but not their asymmetric distributions.An appealing model for symmetry breaking in the early Drosophila wing is that cellular asymmetries of Ft–Ds polarize MT organization and promote the distal transport of Fz–Dsh–Fmi vesicles (Shimada et al., 2001, Harumoto et al., 2010; Matis and Axelrod, 2013; Matis et al., 2014). This would produce an initial bias in Fz–Dsh localization, which would then be amplified by feedback interactions. However, several pieces of evidence have prevented the model from gaining universal acceptance. First, Ds and Fj gradients are oriented in opposite directions with respect to the core PCP proteins in the wing compared with the eye and abdomen. This discrepancy has been rectified with the finding by two independent groups that cells interpret Ft–Ds–Fj gradients differently depending on which of two Pk isoforms is expressed (Ayukawa et al., 2014; Olofsson et al., 2014). Second, the Ft–Ds system can orient PCP independently of the core pathway, and thus the two systems orient polarity in parallel, as opposed to in a single, common pathway (Casal et al., 2006). Third, Ft–Ds mutations affect core PCP orientation only regionally in the wing, which suggests that, if Ft–Ds provides a global bias, other, redundant cues must also exist (Matakatsu and Blair, 2006; Matis et. al., 2014). Finally, the direction of Ft–Ds and core PCP asymmetry diverges late in wing development, where the two systems become completely uncoupled. Intriguingly, the extent of coupling depends on which isoform of Pk is expressed (Merkel et al., 2014). Perhaps the simplest explanation for Ft–Ds function is that it can both transmit polarity information independent of the core system and organize the cytoskeleton to provide an initial bias of core PCP asymmetry, but which mechanism predominates depends on the tissue and developmental stage.

Wnts.

Wnt proteins have long been considered attractive candidates to provide tissue-level polarity cues because Fz and Dsh are primary components of the Wnt–β-catenin signaling pathway. Wnts are secreted glycoproteins that bind to Fz and other receptors, and often display graded expression. In vertebrates, Wnts are clearly important regulators of PCP, but whether they act instructively or permissively is unclear. In zebrafish, Wnt5a and Wnt11 are required for CE movements during gastrulation, but uniform expression of Wnt11 rescues the mutant phenotype, which suggests that it is permissive rather than instructive (Heisenberg et al., 2000; Kilian et al., 2003). Wnt5a is expressed in a gradient along the axis of polarity in the mouse inner ear, where it interacts genetically with Vangl2 in cochlear hair cell orientation (Qian et al., 2007). In the mouse limb, Wnt5a and its atypical receptor Ror2 are required for limb elongation and the asymmetric localization of Vangl2 at the proximal face of converging and extending chondrocytes (Gao et al., 2011). Wnt5a is expressed in a distal-to-proximal gradient, which induces a gradient of Vangl2 phosphorylation. The functional consequences of Vangl2 phosphorylation are unknown but Vangl2 cellular asymmetry appears to be strongest distally, where Wnt5a and Vangl2 phosphorylation levels are highest (Gao et al., 2011).While several studies had argued against the involvement of Wnt proteins in Drosophila PCP (Lawrence et al., 2002; Chen et al., 2008), it was recently discovered that Wingless (Wg) and Wnt4a act redundantly to orient PCP in the wing, particularly near the wing margin (Wu et al., 2013). Misexpression of Wg or Wnt4a reorients wing hair polarity in a pattern reminiscent of Fz loss of function, which suggests that Wnt gradients may orient polarity by antagonizing Fz. Consistently, the ability of Fz and Vang to recruit one another between adjacent cells in culture was inhibited by the addition of Wg or Wnt4a, which suggests that Wnts could provide a polarizing cue by diminishing Fz–Vang interactions at the margin of the wing, where Wnt expression is highest (Wu et al., 2013). However, Wnt4a overexpression also reorients MT alignment, suggesting that Wnts may act as polarity cues thorough an effect on the cytoskeleton (Matis et. al., 2014). Alternatively, Sagner et al. (2012) suggest that Wg orients core PCP indirectly through its effects on wing patterning and growth. Although the evidence for Wnt gradients as global PCP cues is accumulating, the mechanisms by which they regulate core protein levels or activity remain to be elucidated.

Mechanical forces.

Anisotropic mechanical forces that accompany growth and morphogenesis can also provide global polarizing cues. During wing development, PCP reorients in response to extensive morphogenetic changes that elongate the wing along the proximal–distal axis. In early pupal wings, PCP aligns toward the wing margin and then reorients during wing elongation and contraction of the wing hinge (Aigouy et al., 2010). These morphogenetic changes have broad effects on cell behavior, inducing cell elongation, oriented divisions, and cell rearrangements with a concomitant reorientation of PCP. Severing the wing pouch from the hinge blocks cell flows and PCP reorientation, which suggests that the anisotropic tension from hinge contraction drives tissue flow and the reorientation of polarity (Aigouy et al., 2010). Although this model doesn’t explain what initially biases PCP, it does demonstrate how the morphogenetic processes that shape tissues can completely remodel global PCP alignment. This is an attractive model to explain how PCP aligns over very large tissues, like the mammalian skin, where hairs consistently reorient along regions of extensive tissue elongation such as the face, limbs, and ears.

Downstream effectors of PCP: Steering the wheel

If PCP is the cell’s compass, it is also the steering wheel, directing downstream, polarized cell behaviors in response to global directional cues. PCP can polarize a wide range of cell behaviors, which suggests that it can intersect with numerous downstream effectors. We focus here on three examples where the molecular mechanisms linking core PCP to their polarized outputs have recently been elucidated.

Distal positioning of wing hairs.

Each cell of the Drosophila wing blade emits a single actin-rich protrusion from its distal edge. The placement of the wing hair strongly correlates with the position of Fz–Dsh–Fmi, which suggests that core proteins may localize cytoskeletal regulators to distinct positions within the cell (Strutt and Warrington, 2008). On the proximal side, Vang recruits a group of proteins that negatively regulate actin prehair formation: Inturned, Fuzzy, and Fritz (Adler et al., 2004; Strutt and Warrington, 2008). These three proteins regulate Multiple Wing Hairs, a GTPase-binding/formin-homology 3 (GBD/FH3) domain protein thought to repress actin polymerization (Strutt and Warrington, 2008; Yan et al., 2008). This restricts actin nucleation to distal positions within the cell, and in the absence of Multiple Wing Hairs, ectopic actin bundles form across the apical surface (Wong and Adler, 1993). On the distal side, casein kinase 1 γ CK1g/gilamesh is required to further refine prehair nucleation to a single site through a parallel mechanism involving Rab11-dependent vesicle traffic to the site of prehair formation (Gault et al., 2012). Rho and Rho kinase (Drok) have also been implicated in wing hair formation, but their roles are difficult to dissect due to the numerous functions of Rho in cell shape and cell division (Winter et al., 2001; Yan et al., 2009).

Actomyosin contraction and convergent extension (CE).

CE was the first vertebrate process to be linked molecularly to PCP (Wallingford et al., 2000). During CE, mesenchymal cells elongate, form mediolateral-directed protrusions, and intercalate mediolaterally, narrowing the mediolateral axis while simultaneously lengthening the anterior–posterior (A-P) axis (Fig. 3 A; Keller, 2002). Mediolateral polarization, elongation, and intercalation are lost when core PCP components are disrupted, leading to a failure in CE (Tada and Smith, 2000; Wallingford et al., 2000; Goto and Keller, 2002; Jessen et al., 2002). While several PCP-dependent mechanisms have been proposed to mediate CE movements, two recent studies provide direct mechanistic links between asymmetrically localized core PCP components and CE behaviors. In neuroepithelial cells, PCP specifies the localization of myosin to the A-P faces of intercalating cells. Fmi/Celsr1 and Dvl recruit the formin DAAM1 to the A-P junction, which in turn binds and activates PDZ-RhoGEF. This likely activates RhoA and myosin contractility specifically at A-P junctions, resulting in medial-directed cell intercalation and neural plate bending (Nishimura et al., 2012). A similar mechanism was found to drive CE movements of mesenchymal cells during Xenopus gastrulation. In this case, Fritz and Dsh help to localize septins to mediolateral vertices, where they spatially restrict cortical actomyosin contractility and junctional shrinking to A-P cell edges, thus driving cell intercalation (Fig. 3 A; Kim et al., 2010; Shindo and Wallingford, 2014). Together these studies show how asymmetric PCP localization produces collectively polarized cell behaviors through spatial modulation of the cytoskeleton.Open in a separate windowFigure 3.Polarized cell behaviors controlled by PCP. (A) PCP drives convergent extension (CE). CE in vertebrates is driven by mediolateral intercalation, which narrows the mediolateral axis while simultaneously lengthening the A-P axis. Mediolateral intercalation is accompanied by cell polarization and elongation and the formation of mediolateral protrusions, all of which require core PCP function. Pk localizes anteriorly (Ciruna et. al., 2006; Yin et al., 2008), whereas Dsh localizes posteriorly (Yin et. al., 2008). In addition, PCP proteins recruit myosin to A-P cell borders, leading to actomyosin contractility and junctional shrinking. (B) Asymmetric cell division. Drosophila sensory organ precursors (SOPs) divide asymmetrically along the epithelial plane, giving rise to distinct anterior and posterior daughters. Spindle alignment along the A-P axis is PCP dependent. Dsh interacts with Mud/NuMA and the dynein complex posteriorly while Vang links Pins/LGN-Mud/NuMA-dynein on the anterior. This links astral MTs to the A-P cortex, bringing the spindle into register with the A-P axis. (C) Positioning of the kinocilium in the inner ear. The placement of kinocilium in sensory hair cells of the inner ear determines the position of V-shaped stereocilia bundles. Gαi and mPins/LGN localize on the abneural side on the hair cell, where they are required for abneural positioning the MT-based kinocilium. The collective alignment of kinocilia and stereocilia bundles across the epithelium requires the core PCP component Vangl2. Vangl2 (light green) localizes to the abneural side of supporting cells. Whether Fz (dark blue) associates on the opposite face is not yet clear (Ezan et. al., 2013).

Positioning of centrosomes and cilia.

PCP regulates the positioning of MT-based structures including the mitotic spindle and cilia. In Drosophila sensory organ precursors and early zebrafish embryos, PCP controls mitotic spindle orientation along the epithelial plane by interacting with the highly conserved spindle orientation complex, which links astral MTs to the cell cortex through Mud/NuMA-mediated recruitment of the dynein complex (Ségalen et al., 2010). To orient the spindle, posteriorly localized Dsh binds to Mud/NuMA, which recruits the dynein complex and astral MTs to the posterior cortex. On the anterior side, Pins/LGN recruits Mud/NuMA, bringing the spindle into A-P alignment (Fig. 3 B). Similarly, PCP was recently shown to interact with the spindle orientation machinery to position the kinocilium in nondividing cells of the inner ear (Ezan et al., 2013; Tarchini et al., 2013). In vestibular hair cells, Gαi and mPins/LGN localize to the abneural cortex, opposite Vangl2, where they are required for kinocilium positioning and subsequent alignment of stereocilia bundles (Fig. 3 C; Ezan et al., 2013). MT plus ends and dynein also show an abneural bias suggesting that Gαi-mPins/LGN induces pulling on MTs by a similar mechanism that orients the centrosome during spindle orientation. Vangl2 is required for the alignment of Gαi-Pins/LGN crescents between cells, coordinating kinocilia positioning and stereocilia polarity across the tissue (Fig. 3 C; Ezan et al., 2013). Thus the PCP pathway co-opts the spindle orientation machinery to specify not only the division plane but also cilia position in nondividing cells. As PCP is required for asymmetric cilia positioning in a wide range of cell types, including the node (Antic et al., 2010; Borovina et. al., 2010; Song et al., 2010; Hashimoto et. al., 2010), it will be interesting to determine whether this mechanism is conserved.

Concluding remarks

PCP is a fundamental and highly conserved process coordinating a vast number of polarized cell behaviors. While the number of functions ascribed to PCP continues to grow, an understanding of the mechanisms establishing PCP is still far from complete. The development of cellular asymmetry from uniform distributions is not well understood, and will benefit from recent advances in high-resolution, time-lapse imaging with photoconvertible fluorescent tags. Other important issues to resolve include deciphering the structural domains and biochemical interactions mediating intercellular communication, identifying the global cues that orient PCP especially in vertebrates, and deciphering the mechanisms by which complex multicellular structures, like lung branches and hair follicles, are oriented by the PCP machinery.  相似文献   

16.
The concepts of scaling and refractoriness in psychophysical theories of vision     
W. A. van de Grind  J. J. Koenderink  H. A. A. Landman  M. A. Bouman 《Biological cybernetics》1971,8(3):105-122
Summary In the context of the quantum theory of vision scalers, coincidence scalers, adapting coincidence scalers and dead time mechanisms have been used as basic constituents of network models: van de Grind et al. (1970a), Koenderink et al. (1970a, b). The possibilities that these devices offer to construct network models of vision are presently further analysed. First of all a mechanistic analysis is given of the event rate reduction characteristics of dead time boxes. Next the interaction of these devices with scalers is discussed in relation with a number of fluctuation models of vision proposed in the literature. A critical evaluation of these fluctuation models shows an important defect of most of them, viz. that unrealizable detection criteria are postulated. Our reconsideration of this detection problem then leads to the proposal of some specific realizable detectors. An application of the developed theory of mechanisms (machines) to the explanation of the flash detection characteristics of Limulus concludes the paper. Applications of the presented ideas to neural theory and modelling are treated hi a separate paper (van de Grind et al., 1970b) and for applications of the theory to psychophysically oriented visual modelling studies the reader is referred to Koenderink et al. (1970a, b) and van de Grind et al. (1970a).  相似文献   

17.
Genome-Wide Analysis of Yield in Europe: Allelic Effects Vary with Drought and Heat Scenarios     
Emilie J. Millet  Claude Welcker  Willem Kruijer  Sandra Negro  Aude Coupel-Ledru  Stéphane D. Nicolas  Jacques Laborde  Cyril Bauland  Sebastien Praud  Nicolas Ranc  Thomas Presterl  Roberto Tuberosa  Zoltan Bedo  Xavier Draye  Bj?rn Usadel  Alain Charcosset  Fred Van Eeuwijk  Fran?ois Tardieu 《Plant physiology》2016,172(2):749-764
Assessing the genetic variability of plant performance under heat and drought scenarios can contribute to reduce the negative effects of climate change. We propose here an approach that consisted of (1) clustering time courses of environmental variables simulated by a crop model in current (35 years × 55 sites) and future conditions into six scenarios of temperature and water deficit as experienced by maize (Zea mays L.) plants; (2) performing 29 field experiments in contrasting conditions across Europe with 244 maize hybrids; (3) assigning individual experiments to scenarios based on environmental conditions as measured in each field experiment; frequencies of temperature scenarios in our experiments corresponded to future heat scenarios (+5°C); (4) analyzing the genetic variation of plant performance for each environmental scenario. Forty-eight quantitative trait loci (QTLs) of yield were identified by association genetics using a multi-environment multi-locus model. Eight and twelve QTLs were associated to tolerances to heat and drought stresses because they were specific to hot and dry scenarios, respectively, with low or even negative allelic effects in favorable scenarios. Twenty-four QTLs improved yield in favorable conditions but showed nonsignificant effects under stress; they were therefore associated with higher sensitivity. Our approach showed a pattern of QTL effects expressed as functions of environmental variables and scenarios, allowing us to suggest hypotheses for mechanisms and candidate genes underlying each QTL. It can be used for assessing the performance of genotypes and the contribution of genomic regions under current and future stress situations and to accelerate breeding for drought-prone environments.With climate changes, crops will be subjected to more frequent episodes of drought and high temperature that may threaten food security (IPCC, 2014). Reducing the impacts of these effects is an urgent priority that (not exclusively) involves the genetic progress of plant performance under heat and drought stresses (Tester and Langridge, 2010; Lobell et al., 2011). Because hundreds of new genotypes of most cereals are commercialized every year, a generic approach is needed to avoid an endless series of experiments assessing the performances of the newly released genotypes. A systematic exploration of the natural genetic diversity used in breeding can provide information usable for large groups of genotypes. This entails the identification, among the thousands of accessions existing in gene banks, of allelic variants exhibiting specific adaptation traits by addressing three questions: (1) Is there a genetic variability for yield and related traits in dry and hot environments? (2) Can this genetic variability be dissected into the effect of genomic regions (quantitative trait loci, QTLs), and (3) have these genomic regions differential effects depending on environmental conditions (QTL × environment interaction)? Advances in DNA marker analyses and sequencing technologies have decreased the cost of genotyping so the genome of thousands of plants can be densely characterized (Langridge and Fleury, 2011). Genome-wide association study (GWAS) allows associations of phenotypic traits with causal polymorphisms (Zhu et al., 2008) but, in our analysis, needs to be fine-tuned for plant responses to climatic scenarios associated with climate change. In particular, several options exist for the experimental strategy. (1) The comparison of genotype performances can be addressed in controlled infrastructures that simulate conditions in 2050—for instance, in phenotyping platforms (Tardieu and Tuberosa, 2010; Fiorani and Schurr, 2013) or in fields with managed environments (Salekdeh et al., 2009; Bishop et al., 2015). However, these possibilities do not address the diversity of environmental scenarios faced by plants in current and future conditions. (2) Panels of genotypes can be analyzed in a network of field experiments, resulting in the association of performances with genomic regions depending on environmental indices that best account for QTL×E interaction (Vargas et al., 2006; Malosetti et al., 2013; Bouffier et al., 2015). However, each network of experiments results in its own set of indices that cannot be easily compared between studies, nor extended to a whole geographic region.We propose here an approach that consists in utilizing the current year-to-year and site-to-site climatic variability for genetic analyses of plant performance in current climatic scenarios and in those predicted for the future. It consists of (1) clustering current and future environmental conditions into a limited number of scenarios as experienced by the studied crop; (2) performing a series of field experiments for a collection of scenarios across Europe; (3) assigning individual experiments to scenarios according to environmental conditions measured in each field experiment; and (4) analyzing the genetic variation for plant performances as a function of environmental scenarios. Here, we address these four steps for maize (Zea mays L.) in Europe. Maize was chosen as a case study because it is a C4 species in which the increase of CO2 has limited effect on photosynthesis.(1) The first step has been performed by running crop simulations over a large range of sites over tens of years and then clustering the simulated time courses of environmental variables into a limited number of environmental scenarios at key phenological stages of the crop (Chapman et al., 2000; Chenu et al., 2011). To address the case of maize grown in Europe, we have used the drought scenarios defined by Harrison et al., (2014) based on 55 European sites over 35 years. We have used the dataset collected in their paper to also identify three scenarios of temperature during the maize cropping cycle under current conditions. Future conditions have been simulated by using the model LARS-WG (Semenov and Stratonovitch, 2010).(2) The second step consisted in performing field experiments with a panel of genotypes over a range of conditions. This was done in 29 field experiments (defined as combinations of site × year × watering regime), in which a panel of 244 maize hybrids was analyzed along a climatic transect from west to east Europe, plus one experiment in Chile. This panel, genotyped with 515 000 single nucleotide polymorphism (SNP) markers, maximized the genetic variability in the dent maize group while restricting the range of flowering time to 10 d in order to avoid confounding the effects of phenology with intrinsic responses to drought and heat. It included first-cycle lines derived from historical landraces and more recent lines created by public institutions and breeding companies.(3) The third step ascribed each experiment to an environmental scenario defined in step 1. This required full environmental characterization of each individual experiment. We expected that the proportion of experiments belonging to each environmental scenario might appreciably differ from those calculated over 55 sites × 35 years. Hence, this step allowed us to give a weight to each experiment according to environmental conditions in this experiment and to frequencies of environmental scenarios. It was therefore not a simple classification of experiments of the network as performed by other groups (Vargas et al., 2006; Malosetti et al., 2013; Bouffier et al., 2015).(4) The fourth step consisted in evaluating the genetic variability of yield and of related variables within each climatic scenario, in identifying genomic regions associated with these traits in each scenario and in relating allelic effects to measured environmental conditions. Indeed, associations between markers and yield under stress pose a specific challenge because every significant marker may have opposite allelic effects depending on the timing and severity of drought or heat stresses (Vargas et al., 2006; Boer et al., 2007; Collins et al., 2008; Tardieu, 2012). The analysis of a network of dry field experiments has shown that a given allele at a QTL can have a markedly positive effect in one category of experiments, a markedly negative effect in another category, and nearly no effect in half of experimental fields (Bonneau et al., 2013). Hence, we have first performed single-environment GWAS that allows identification of QTLs strongly associated with specific experiments and multi-environment GWAS that allows identification of QTLs with both main effect and QTL × E effects (Boer et al., 2007; Maccaferri et al., 2008; Malosetti et al., 2008a; Maccaferri et al., 2016). We have then analyzed the effects of QTL alleles conditional on scenarios and measured environmental conditions.We could, in this way, estimate the frequencies of positive, negative, or null effects for each QTL in each climatic scenario, depending on measured environmental conditions in each field. This resulted in a pattern of QTL effects as a function of scenarios, environmental variables (e.g. temperature versus evaporative demand versus soil water potential) and traits (e.g. flowering time versus grain number versus grain size). We have deduced from these patterns hypotheses for the mechanisms underlying the QTLs, thereby helping in the selection of candidate genes among the small number of possible genes close to causal polymorphisms. Hence, this work aimed to bring together GWAS and ecophysiological analyses for modeling and providing biological/ecological interpretation of conditional QTL effects associated to ranges of soil water deficit, evaporative demand, and air temperature across Europe in current and future climatic scenarios.  相似文献   

18.
States of high conductance in a large-scale model of the visual cortex     
Shelley M  McLaughlin D  Shapley R  Wielaard J 《Journal of computational neuroscience》2002,13(2):93-109
This paper reports on the consequences of large, activity dependent, synaptic conductances for neurons in a large-scale neuronal network model of the input layer 4C of the Macaque primary visual cortex (Area V1). This high conductance state accounts for experimental observations about orientation selectivity, dynamics, and response magnitude (D. McLaughlin et al. (2000) Proc. Natl. Acad. Sci. USA 97: 8087–8092), and the linear dependence of Simple cells on visual stimuli (J. Wielaard et al. (2001) J. Neuroscience 21: 5203–5211). The source of large conductances in the model can be traced to inhibitory corticocortical synapses, and the model's predictions of large conductance changes are consistent with recent intracellular measurements (L. Borg-Graham et al. (1998) Nature 393: 369–373; J. Hirsch et al. (1998) J. Neuroscience 15: 9517–9528; J.S. Anderson et al. (2000) J. Neurophysiol. 84: 909–926). During visual stimulation, these conductances are large enough that their associated time-scales become the shortest in the model cortex, even below that of synaptic interactions. One consequence of this activity driven separation of time-scales is that a neuron responds very quickly to temporal changes in its synaptic drive, with its intracellular membrane potential tracking closely an effective reversal potential composed of the instantaneous synaptic inputs. From the effective potential and large synaptic conductance, the spiking activity of a cell can be expressed in an interesting and simplified manner, with the result suggesting how accurate and smoothly graded responses are achieved in the model network. Further, since neurons in this high-conductance state respond quickly, they are also good candidates as coincidence detectors and burst transmitters.  相似文献   

19.
The derivation of nerve signals from contrast flash data     
J. Walraven 《Biological cybernetics》1980,38(1):23-29
This paper presents a new analysis of the contrast flash data of Alpern et al. (1970a-d). It was prompted by the criticism of Wandell (1976) who pointed out that Alpern et al., main conclusion, i.e. that the inhibitory signal N *() elicited by the contrast flash () takes the form , would imply an unrealistic excitatory photo response. The present analysis shows the data to be consistent with an inhibitory signal of the form .  相似文献   

20.
Homing in the Digger Wasp Bembix rostrata (Hymenoptera,Sphecidae) in Relation to Sex and Stage1     
Jan Teng  Hermann Schne  Jerzy Chmurzy&#x;ski 《Ethology : formerly Zeitschrift fur Tierpsychologie》1990,86(1):47-56
In the digger wasp Bembix rostrata in males and females of different stages the homing rates and times after release from different distances were investigated.
  • 1 The homing rate decreased with increase of distance (up to 3000 m).
  • 2 In females the homing rate increased with progressing stage, from emerging over digging to provisioning.
  • 3 Numbers for homing times followed the same order: highest in emerging, medium in digging and lowest in provisioning females.
  • 4 Males showed similar homing rates as digging females. No significant differences between age groups could be found.
  • 5 The results indicate an effect of sex and stage on the homing capacity. Differences may be traced back to motivation (provisioning females) and lack of experience (emerging females). Landmark orientation, which may include particular search strategies, is a probable orientation mechanism.
  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号