Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape

The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.     

Tor complex 1 controls telomere length by affecting the level of Ku

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeric DNA is synthesized by telomerase, which is expressed only at the early stages of development [ [1] and [2] ]. To become malignant, any cell has to be able to replenish telomeres [3]. Thus, understanding how telomere length is monitored has significant medical implications, especially in the fields of aging and cancer. In yeast, telomerase is constitutively active. A large network of genes participates in controlling telomere length [ [4] , [5] , [6] , [7] and [8] ]. Tor1 and Tor2 (targets of rapamycin [9]) are two similar kinases that regulate cell growth [10]. Both can be found as part of the TOR complex 1 (TORC1 [11]), which coordinates the response to nutrient starvation and is sensitive to rapamycin [12]. The rapamycin-insensitive TOR complex 2 (TORC2) contains only Tor2 and regulates actin cytoskeleton polarization [13]. Here we provide evidence for a role of TORC1 in telomere shortening upon starvation in yeast cells. The TORC1 signal is transduced by the Gln3/Gat1/Ure2 pathway, which controls the levels of the Ku heterodimer, a telomere regulator. We discuss the potential implications for the usage of rapamycin as a therapeutic agent against cancer and the effect that calorie restriction may have on telomere length.     

A selective difference between human Y-chromosomal DNA haplotypes

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] and [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] and [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] and [11] and as many as 5% of males are infertile [11] and [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.     

Elevation of superoxide dismutase increases acoustic trauma from noise exposure

The generation of superoxide has been implicated as a cause of cochlear damage from excessive noise. Cu/Zn superoxide dismutase (SOD1) generally will protect against superoxide-mediated tissue injury but protection by this enzyme against noise trauma is controversial. This study assessed auditory function in C57BL/6 mice overexpressing SOD1 or treated with lecithinized SOD1 (PC-SOD1). Noise exposure caused significantly higher threshold shifts in PC-SOD1-treated animals than physiological saline-treated animals. Cochlear tissues of PC-SOD1-treated animals exhibited significant elevation of the levels in the SOD activity, not in the catalase activity, in comparison with those of saline-treated animals. Likewise, transgenic mice overexpressing SOD1 tended to suffer higher threshold shifts than nontransgenic littermates from noise exposure. The findings indicate that increasing SOD1 enhances auditory dysfunction following noise exposure.     

Actin depolymerization-based force retracts the cell rear in polarizing and migrating cells

In migrating cells, the relative importance of myosin II contractility for cell rear retraction varies [ [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] and [12] ]. However, in myosin II-inhibited polarizing cells, actin organization is compromised [ [13] , [14] , [15] , [16] , [17] and [18] ]; thus it remains unclear whether myosin II is simply required for correct actin arrangement or also directly drives rear retraction [9]. Ascaris sperm cells lack actin and associated motors, and depolymerization of major sperm protein is instead thought to pull the cell rear forward [ [19] and [20] ]. Opposing views exist on whether actin could also have this function [ [19] and [20] ] and has not been directly experimentally sought. We probe function at high temporal resolution in polarizing fibroblasts that establish migration by forming the cell rear first [ [9] , [15] and [21] ]. We show that in cells with correctly organized actin, that actin filament depolymerization directly drives retraction of the rear margin to polarize cells and spatially accounts for most cell rear retraction during established migration. Myosin II contractility is required early, to form aligned actin bundles that are needed for polarization, and also later to maintain bundle length that ensures directed protrusion at the cell front. Our data imply a new mechanism: actin depolymerization-based force retracts the cell rear to polarize cells with no direct contribution from myosin II contractility.     

Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [ [3] and [4] ]; its overexpression enhances cellular motility and metastatic activity [ [5] , [6] , [7] and [8] ]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [ [9] and [10] ], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.     

Superoxide dismutase: the balance between prevention and induction of oxidative damage

Cu,Zn-superoxide dismutase (SOD1) has been shown to be effective in several free radical mediated diseases, although some studies have pointed toward SOD1 toxicity at a high concentrations. In the present study, the balance between prevention and induction of damage by SOD1 has been investigated both in vitro and in vivo. In vitro superoxide was generated using xanthine/xanthine oxidase. In vivo superoxide was generated using the redox cycling compound doxorubicin. Furthermore, we determined the pharmacokinetics of lecithinized SOD1 (PC-SOD) in order to compare the results obtained in vivo with those obtained in vitro. It was found that in vitro high concentrations of SOD1 induce hydroxylation of coumarin 3-carboxylic acid (3-CCA). This could be caused by a peroxidative action of SOD1 or formation of the highly reactive hydroxyl radicals. Any signs of toxicity are absent in vivo because these concentrations are not reached. It can be concluded that SOD1 possesses a large therapeutic window and application of SOD1 or its derivatives for strengthening the body's defenses against oxidative stress in a variety of pathologies seems safe.     

Effect of lecithinized superoxide dismutase (PC-SOD) on experimental pulmonary metastasis in mice.

The inhibitory effect of lecithinized superoxide dismutase (PC-SOD) on pulmonary metastasis in mice was investigated. In an experimental pulmonary metastasis model employing Meth A-T cells, significant and dose-dependent inhibition was observed after i.v. pre-administration of PC-SOD. Unmodified SOD (U-SOD) was also effective, but a 10-times higher dose was necessary to be significant. The pulmonary accumulation of Meth A-T cells labeled with 5-[125I]iodo-2'-deoxyuridine was not reduced by either PC-SOD or U-SOD, and neither of the compounds decreased pulmonary MPO activity. However, PC-SOD increased pulmonary SOD activity for longer, compared with U-SOD. In vitro addition of PC-SOD dose-dependently suppressed the growth of Meth A-T cells, while U-SOD had little effect. The combination of PC-SOD and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitric oxide (NO)-generating agent, had an additive effect. It was also found that PC-SOD prevented a decrease of pulmonary NOx level following tumor cell inoculation. It was concluded that PC-SOD possessed antimetastatic activity, and its potency was superior to that of U-SOD. These results suggest that PC-SOD may prevent the excessive formation of oxygen radicals and peroxynitrite (ONOO-) which cause cell damage and facilitate tumor metastasis.     

Liposome membrane can act like molecular and metal chaperones for oxidized and fragmented superoxide dismutase

A mechanism for liposome-recruited activity of oxidized and fragmented superoxide dismutase (Fr.-SOD) [Tuan LQ, Umakoshi H, Shimanouchi T, Kuboi R. Liposome-recruited activity of oxidized and fragmented superoxide dismutase. Langmuir 2008;24:350–4] was further investigated, focusing on the secondary structure of Fr.-SOD. Liposome membrane was found to assist the conformational change of Fr.-SOD and reactivate the enzymatic activity, like molecular and metal chaperones. The loss of SOD activity and its secondary structure was observed during 6 h oxidation in 2 mM hydrogen peroxide. The contents of the α-helix and β-sheet structures in the oxidized and fragmented SOD (2 μM) were increased only in the presence of 10 μM Cu²⁺ and Zn²⁺ together, or in the presence of 2 mM POPC liposomes. The mixture of all of these elements (fragmented SOD and POPC liposomes with Cu²⁺ and Zn²⁺) gave not only the increase of the α-helix and β-sheet contents but also the mediation of the high SOD-like activity.     

Optical reversal of halothane-induced immobility in C. elegans

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [ [1] and [2] ]. Genetic studies in Caenorhabditis elegans [ [3] and [4] ], Drosophila [ [3] and [5] ], and mice [ [6] , [7] , [8] and [9] ] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K⁺ [ [10] , [11] , [12] and [13] ] or permeable to Na⁺ [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.     

Intravenous administration of superoxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a rat model of adjuvant arthritis.

Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired.     

Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.     

Superiority of PC-SOD to other anti-COPD drugs for elastase-induced emphysema and alteration in lung mechanics and respiratory function in mice

Bronchodilators (such as ipratropium bromide), steroids (such as fluticasone propionate), and newly developed anti-inflammatory drugs (such as roflumilast) are used for patients with chronic obstructive pulmonary disease (COPD). We recently reported that lecithinized superoxide dismutase (PC-SOD) confers a protective effect in mouse models of COPD. We here examined the therapeutic effect of the combined administration of PC-SOD with ipratropium bromide on pulmonary emphysema and compared the effect of PC-SOD to other types of drugs. The severity of emphysema in mice was assessed by various criteria. Lung mechanics (elastance) and respiratory function (ratio of forced expiratory volume in the first 0.05 s to forced vital capacity) were assessed. Administration of PC-SOD by inhalation suppressed elastase-induced pulmonary emphysema, alteration of lung mechanics, and respiratory dysfunction. The concomitant intratracheal administration of ipratropium bromide did not alter the ameliorating effects of PC-SOD. Administration of ipratropium bromide, fluticasone propionate, or roflumilast alone did not suppress the elastase-induced increase in the pulmonary level of superoxide anion, pulmonary inflammatory response, pulmonary emphysema, alteration of lung mechanics, or respiratory dysfunction as effectively as did PC-SOD. PC-SOD, but not the other drugs, showed a therapeutic effect even when the drug was administered after the development of emphysema. PC-SOD also suppressed the cigarette smoke-induced pulmonary inflammatory response and increase in airway resistance. Based on these results, we consider that the inhalation of PC-SOD would be therapeutically beneficial for COPD.     

Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 °C and 4 °C, was investigated in living cells. Compared with 37 °C, the uptake of R8-liposomes was only slightly reduced at 4 °C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 °C. When introduced at 4 °C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 °C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 °C, was less prominent compared with those introduced at 37 °C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.     

Spectroscopic features of cytochrome P450 reaction intermediates

Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2] and [3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV–vis absorption spectra of the reduced CO-saturated state [4] and [5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle.     

Shaping fission yeast cells by rerouting actin-based transport on microtubules

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [ [2] , [3] and [4] ]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.

Video Abstract

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20.

Acquiring "the Knowledge" of London's layout drives structural brain changes     

Woollett K  Maguire EA 《Current biology : CB》2011,21(24):2109-2114

The last decade has seen a burgeoning of reports associating brain structure with specific skills and traits (e.g., [ [1] , [2] , [3] , [4] , [5] , [6] , [7] and [8] ]). Although these cross-sectional studies are informative, cause and effect are impossible to establish without longitudinal investigation of the same individuals before and after an intervention. Several longitudinal studies have been conducted (e.g., [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] and [18] ]); some involved children or young adults, potentially conflating brain development with learning, most were restricted to the motor domain, and all concerned relatively short timescales (weeks or months). Here, by contrast, we utilized a unique opportunity to study average-IQ adults operating in the real world as they learned, over four years, the complex layout of London's streets while training to become licensed taxi drivers. In those who qualified, acquisition of an internal spatial representation of London was associated with a selective increase in gray matter (GM) volume in their posterior hippocampi and concomitant changes to their memory profile. No structural brain changes were observed in trainees who failed to qualify or control participants. We conclude that specific, enduring, structural brain changes in adult humans can be induced by biologically relevant behaviors engaging higher cognitive functions such as spatial memory, with significance for the “nature versus nurture” debate.     

Acquiring "the Knowledge" of London's layout drives structural brain changes

The last decade has seen a burgeoning of reports associating brain structure with specific skills and traits (e.g., [ [1] , [2] , [3] , [4] , [5] , [6] , [7] and [8] ]). Although these cross-sectional studies are informative, cause and effect are impossible to establish without longitudinal investigation of the same individuals before and after an intervention. Several longitudinal studies have been conducted (e.g., [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] and [18] ]); some involved children or young adults, potentially conflating brain development with learning, most were restricted to the motor domain, and all concerned relatively short timescales (weeks or months). Here, by contrast, we utilized a unique opportunity to study average-IQ adults operating in the real world as they learned, over four years, the complex layout of London's streets while training to become licensed taxi drivers. In those who qualified, acquisition of an internal spatial representation of London was associated with a selective increase in gray matter (GM) volume in their posterior hippocampi and concomitant changes to their memory profile. No structural brain changes were observed in trainees who failed to qualify or control participants. We conclude that specific, enduring, structural brain changes in adult humans can be induced by biologically relevant behaviors engaging higher cognitive functions such as spatial memory, with significance for the “nature versus nurture” debate.