Cell Biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.     

2.

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells     

P. I. Liu  K. C. Poon  L. Crook  S. S. Liu  C. C. Hong 《Cryobiology》1979,16(6):513-516

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates

a. Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells^a

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates

a. Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells^a

Introducing Titratable Water to All-Atom Molecular Dynamics at Constant?pH

Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pK_a values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λ_i} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pK_a values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:

• 1.Loss of a proton to give a negatively charged hydroxide,

H₂O ? OH^? + H⁺, (1)or

• 2.Gain of a proton to give a positively charged hydronium,

H₂O + H⁺ ? H₃O⁺.(2)We now couple the titration of hydroxide (Eq. 1) with that of an acidic site of the solute in the CpHMD simulation,HA+OH−⇌KaA−+H2O.(3)The use of hydronium is avoided here to prevent a potential artifact due to prolonged attraction with A⁻. Analogously, we couple the titration of hydronium (Eq. 2) with that of a basic site,BH++H2O⇌KbH3O++B.(4)Thus, effectively, a proton is transferred between the solute and solvent. However, we should note that in CpHMD simulations, titratable protons are represented by covalently attached dummies (2,4). Through varying the atomic charges and van der Waals interactions, they are seen by other atoms in the protonated state but not in the unprotonated state (see Table S1 in the Supporting Material). Furthermore, the solution proton concentration is implicitly modeled through a free energy term (2,4).In CpHMD, the reference potential of mean force (PMF) for titration is that of the model compound (blocked single amino acid in water) along λ (2,4). In the presence of cotitrating water molecules, it is necessary to add the PMF for the conversion of water to hydroxide or hydronium. One-nanosecond NPT simulations at ambient pressure and temperature were performed to calculate the average force, 〈dU/d,θ〉 at given θ-values, which are related to λ by λ = sin² θ (see Fig. S1 in the Supporting Material). Thermodynamic integration was then applied to calculate the PMF. We found that the average force can be accurately fit when assuming the PMF is quadratic in λ (Fig. 1). The same applies to the PMFs for titration of models Asp, Glu, and His. After testing on the titration of model compounds (see Table S2), we performed 10-ns all-atom CpHMD simulations with the pH replica-exchange protocol for three proteins: HP36, BBL and HEWL (see the Supporting Material for details). Most of the calculated pK_a values were converged in 10 ns per replica (see Fig. S3). Results are summarized in Fig. S4. Based on the 10-ns data, the root-mean-square (RMS) and average absolute errors are 0.9 and 0.7 pH units, respectively, while the largest absolute error is 2.5 (Glu³⁵ of HEWL). Linear regression of the calculation versus experiment gives R² of 0.8 and slope of 1.2.Open in a separate windowFigure 1Average force and potential of mean force for converting a water molecule to hydroxide (A) and hydronium. (B) (Data points) Average forces. (Dashed curves) Best fits using a linear function, 2A(λ − B). (Solid curves) Corresponding potential of mean force.

Table 1

Calculated and experimental pK_a values of three proteins

Demonstrated and inferred metabolism associated with cytosolic lipid droplets

Cytosolic lipid droplets were considered until recently to be rather inert particles of stored neutral lipid. Largely through proteomics is it now known that droplets are dynamic organelles and that they participate in several important metabolic reactions as well as trafficking and interorganellar communication. In this review, the role of droplets in metabolism in the yeast Saccharomyces cerevisiae, the fly Drosophila melanogaster, and several mammalian sources are discussed, particularly focusing on those reactions shared by these organisms. From proteomics and older work, it is clear that droplets are important for fatty acid and sterol biosynthesis, fatty acid activation, and lipolysis. However, many droplet-associated enzymes are predicted to span a membrane two or more times, which suggests either that droplet structure is more complex than the current model posits, or that there are tightly bound membranes, particularly derived from the endoplasmic reticulum, which account for the association of several of these proteins.Cytosolic lipid droplets, originally thought to be simply coalesced neutral lipids waiting for lipolysis at metabolic demand, are now known to be considerably more complicated both structurally and functionally. There is general agreement that droplets are comprised of a core of neutral lipids, principally triglycerides and steryl esters, surrounded by a leaflet of phospholipids into which are embedded a specific subset of cellular proteins, the most abundant of which are members of the PAT family (see below) in animal cells (1). However, this model is probably too simple; there is evidence from physical probes of droplets isolated from yeast mutants unable to synthesize triglycerides or steryl esters that these two molecular families are partially segregated within the core, with thin shells of steryl esters forming concentric hollow spheres around an inner core composed principally of triglycerides (2).The next layer of complexity is the functional inhomogeneity of droplets. Subsets of droplets within the same cells exist with different populations of PAT proteins, differentiating among different sizes, ages, and levels of metabolic activity (3, 4). Perhaps most surprisingly, droplets may be comprised, at least in some cases, not of the layered core-phospholipid shell architecture at all but a knot of tightly woven endoplasmic reticulum (ER) surrounded by secreted neutral lipid, itself encased with a single leaflet. Such a model is based on electron microscopic thin sections (5), freeze fracture-immunogold evidence (6), immunohistochemical studies of ER luminal proteins within the droplet (7), and the identification of these proteins, notably ER chaperones, in several proteomic studies. Although certainly, such a complex structure must obey physical laws governing aqueous interactions with hydrophobic lipids and artifacts in processing for electron microscopy do occur, it may be best at present to keep an open mind and consider that droplets may not have the same structure among tissues and that they may take multiple physical forms in rapid order as they dynamically perform their functions.What are these functions? The most obvious one is lipid metabolism, namely the biogenesis and breakdown of the neutral lipids contained within the droplet. Although this conclusion predates proteomic studies (8), these recent studies have revealed the breadth and conservation of metabolic reactions that occur at or near the droplet surface, the subject of this review. Moreover, proteomics has demonstrated the surprising fact that droplets are likely to be very active in organellar communication because they are replete in rab proteins and other trafficking molecules. Our knowledge from proteomic studies of droplet trafficking and communication is discussed separately in this thematic review series.A major caveat must be kept in mind when evaluating droplet proteomics data: besides droplet trafficking through transient interactions with vesicles or target organelles such as early endosomes (9), droplets make extensive, tight, and long-lasting synapses with the endoplasmic reticulum, mitochondria, and peroxisomes (10, 11). The fact that ER, mitochondrial, peroxisomal, and a few plasma membrane proteins are found with such high frequency in the droplet proteome probably reflects these tight interorganellar interactions, perhaps similar to the mitochondrially associated membranes (MAMs) that link mitochondria with ER (12). The molecular basis for droplet-mediated synapses are not yet known. Besides the frequent occurrence of specific nondroplet organelle proteins in the droplet proteome, adventitious contamination of droplets is unlikely in view of the unique density of droplets that allow their flotation to the top of aqueous buffers and density gradients after centrifugation while all other cell components sink (which also permits several washes with high recovery), and the nonrandom coisolation of subsets of proteins from other organelles, such as the β-oxidation peroxisomal enzymes (10), which suggests specialized regions for metabolically-productive droplet interactions at the synapses.Droplet-ER interactions are a special case; it is the rule rather than the exception that enzymes of lipid metabolism that are found in the droplet proteome are also found to varying extents in the ER. This has been well documented in yeast through genome-wide green fluorescent protein (GFP)-tagging (13, 14). Erg6p, an enzyme in the latter part of the ergosterol biosynthetic pathway, is the only droplet protein in the pathway with a near-exclusive droplet localization in yeast; Erg1p, Erg7p, and Erg 27p are dually localized, and the pattern changes depending on metabolic state. Whether this general rule is specific for yeast, in which droplets remain on the ER surface (15), is not yet clear. However, several examples already exist in mammalian cells: cytochrome b5 reductase (DT diaphorase) and various sterol dehydrogenases (see 12).

TABLE 1.

Metabolic functions of droplets as revealed by proteomics

Hyperosmotic injury in mammalian cells. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures.

Correlated studies on volume distributions and cation (Na⁺ and K⁺) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 mOsm in both the presence and absence of DMSO (5–20%; $\text{w}\text{v}$). The effects superimposed by ouabain (10^?2–10^?4m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the

TABLE 2. Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension

     

8.

The hippo pathway     

Harvey KF  Hariharan IK 《Cold Spring Harbor perspectives in biology》2012,4(8):a011288

     

9.

ALGAL RESPONSE TO NUTRIENT ENRICHMENT IN FORESTED OLIGOTROPHIC STREAM1     

Annelies J. Veraart  Anna M. Romaní  Elisabet Tornés  Sergi Sabater 《Journal of phycology》2008,44(3):564-572

Nutrient input in streams alters the density and species composition of attached algal communities in open systems. However, in forested streams, the light reaching the streambed (rather than the local nutrient levels) may limit the growth of these communities. A nutrient‐enrichment experiment in a forested oligotrophic stream was performed to test the hypothesis that nutrient addition has only minor effects on the community composition of attached algae and cyanobacteria under light limitation. Moderate nutrient addition consisted of increasing basal phosphorus (P) concentrations 3‐fold and basal nitrogen (N) concentrations 2‐fold. Two upstream control reaches were compared to a downstream reach before and after nutrient addition. Nutrients were added continuously to the downstream reach for 1 year. Algal biofilms growing on ceramic tiles were sampled and identified for more than a year before nutrient addition to 12 months after. Diatoms were the most abundant taxonomic group in the three stream reaches. Nutrient enrichment caused significant variations in the composition of the diatom community. While some taxa showed significant decreases (e.g., Achnanthes minutissima, Gomphonema angustum), increases for other taxa (such as Rhoicosphenia abbreviata and Amphora ovalis) were detected in the enriched reach (for taxonomic authors, see Table 2 ). Epiphytic and adnate taxa of large size were enhanced, particularly during periods of favorable growth conditions (spring). Nutrients also caused a change in the algal chl a, which increased from 0.5–5.8 to 2.1–10.7 μg chl · cm^?2. Our results indicate that in oligotrophic forested streams, long‐term nutrient addition has significant effects on the algal biomass and community composition, which are detectable despite the low light availability caused by the tree canopy. Low light availability moderates but does not detain the long‐term tendency toward a nutrient‐tolerant community. Furthermore, the effects of nutrient addition on the algal community occur in spite of seasonal variations in light, water flow, and water chemical characteristics, which may confound the observations.

Table 2. Percent abundances of the most frequent taxa in three reaches of the Fuirosos stream. U1 and U2 untreated; E, enriched both in the periods before (bef) and after (aft) the enrichment of the E reach. Acronyms identifying the taxa are indicated.

Osmolality mOsm	Exposure (min)								Hypertonic agent
					NaCl				Sucrose
		$\text{V}{}^{\mathrm{a}}$	Na+	K+	$\text{S}{}^{\mathrm{b}}$	V	Na+	K+	S
1000	60 or less	Small	High	High	High	Normal	Low	High	High
1500–2000	60 or less	Small	High	Low	Low	Normal	Low	High	High
2000 or over	60 or more	Small or largec	High	Very low	Very low	Small	Very low	Very low	Very low

		U1‐bef	U1‐aft	U2‐bef	U2‐aft	E‐bef	E‐aft
Achnanthes biasolettiana Grunow	ABIA	1.1	1.2	0.4	0.1	5.4	0.7
Achnanthes lanceolata (Bréb.) Grunow	ALAN	7.2	1.3	5.7	7.1	7.3	2.2
Achnanthes minutissima Kütz.	AMIN	56.2	55.0	81.2	71.4	52.2	34.5
Achnanthes lanceolata v. frequentissima Lange‐Bert.	ALFR	0.0	0.1	0.1	0.9	1.0	0.0
Amphora inariensis Krammer	AINA	1.9	2.0	0.3	0.1	1.0	1.4
Amphora ovalis (Kütz.) Kütz.	AOVA	0.0	0.0	0.0	0.0	0.0	1.3
Amphora pediculus (Kütz.) Grunow	APED	0.9	2.2	0.1	0.6	3.3	1.3
Cocconeis pediculus Ehrenb.	CPED	0.1	0.2	0.0	0.1	0.2	1.7
Cocconeis placentula Ehrenb.	CPLA	13.7	20.3	1.8	8.4	12.3	32.4
Cymbella silesiaca Bleisch in Rabenh.	CSLE	0.0	0.2	0.0	0.1	0.0	0.1
Diploneis oblongella (Nägeli) Cleve‐Euler	DOBL	0.6	0.0	0.9	0.2	0.0	0.0
Fragilaria capucina var. gracilis (Øestrup) Hustedt	FCGP	0.3	1.0	0.1	0.0	0.1	3.5
Fragilaria capucina var. capitellata (Grunow) Lange‐Bert.	FCCP	0.0	0.2	0.0	0.1	0.4	0.6
Fragilaria ulna (Nitzsch) Lange‐Bert.	FULN	0.2	1.1	0.1	0.1	0.0	1.4
Gomphonema angustatum (Kütz.) Rabenh.	GADI	1.6	0.6	1.6	1.8	1.0	0.8
Gomphonema angustum C. Agardh	GANT	0.2	0.1	0.6	1.2	1.4	0.1
Gomphonema minutum (C. Agardh) C. Agardh	GMIN	0.2	0.0	0.3	0.1	0.3	0.5
Gomphonema pumilum (Grunow) E. Reichardt et Lange‐Bert.	GPUM	1.7	0.0	2.0	1.4	1.1	0.0
Meridion circulare (Grev.) C. Agardh	MCIR	0.0	0.1	1.5	1.7	0.4	0.2
Navicula antonii Lange‐Bert.	NANT	0.8	0.1	0.1	0.2	0.8	0.2
Navicula accomoda Hust.	NARB	0.0	0.0	0.0	0.0	0.0	0.0
Navicula capitatoradiata H. Germ.	NCPR	0.3	0.0	0.1	0.1	0.0	0.3
Navicula cryptocephala Kütz.	NCRY	0.5	0.1	0.1	0.3	0.5	0.2
Nitzschia linearis (C. Agardh) W. Sm.	NLIN	0.2	0.0	0.0	0.2	0.0	0.1
Nitzschia palea (Kütz.) W. Sm.	NPAL	0.0	0.0	0.3	0.2	0.5	0.2
Reimeria sinuata (W. Greg.) Kociolek et Stoermer	RSIN	3.4	2.0	0.6	1.2	4.9	2.8
Rhoicosphenia abbreviata (C. Agardh) Lange‐Bert.	RABB	8.1	5.0	0.2	0.4	3.6	9.9

Citing Literature

Volume 44 , Issue 3 June 2008

Pages 564-572     

ALGAL RESPONSE TO NUTRIENT ENRICHMENT IN FORESTED OLIGOTROPHIC STREAM1

Nutrient input in streams alters the density and species composition of attached algal communities in open systems. However, in forested streams, the light reaching the streambed (rather than the local nutrient levels) may limit the growth of these communities. A nutrient‐enrichment experiment in a forested oligotrophic stream was performed to test the hypothesis that nutrient addition has only minor effects on the community composition of attached algae and cyanobacteria under light limitation. Moderate nutrient addition consisted of increasing basal phosphorus (P) concentrations 3‐fold and basal nitrogen (N) concentrations 2‐fold. Two upstream control reaches were compared to a downstream reach before and after nutrient addition. Nutrients were added continuously to the downstream reach for 1 year. Algal biofilms growing on ceramic tiles were sampled and identified for more than a year before nutrient addition to 12 months after. Diatoms were the most abundant taxonomic group in the three stream reaches. Nutrient enrichment caused significant variations in the composition of the diatom community. While some taxa showed significant decreases (e.g., Achnanthes minutissima, Gomphonema angustum), increases for other taxa (such as Rhoicosphenia abbreviata and Amphora ovalis) were detected in the enriched reach (for taxonomic authors, see Table 2 ). Epiphytic and adnate taxa of large size were enhanced, particularly during periods of favorable growth conditions (spring). Nutrients also caused a change in the algal chl a, which increased from 0.5–5.8 to 2.1–10.7 μg chl · cm^?2. Our results indicate that in oligotrophic forested streams, long‐term nutrient addition has significant effects on the algal biomass and community composition, which are detectable despite the low light availability caused by the tree canopy. Low light availability moderates but does not detain the long‐term tendency toward a nutrient‐tolerant community. Furthermore, the effects of nutrient addition on the algal community occur in spite of seasonal variations in light, water flow, and water chemical characteristics, which may confound the observations.

Table 2. Percent abundances of the most frequent taxa in three reaches of the Fuirosos stream. U1 and U2 untreated; E, enriched both in the periods before (bef) and after (aft) the enrichment of the E reach. Acronyms identifying the taxa are indicated.

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

     

11.

Endoplasmic Reticulum Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway     

Vladimir Denic  Volker D?tsch  Irmgard Sinning 《Cold Spring Harbor perspectives in biology》2013,5(8)

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their carboxyl terminus. Many of these tail-anchored (TA) proteins are posttranslationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the guided-entry of TA protein insertion (GET) pathway. In recent years, most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway-targeting factor that uses nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pretargeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.The mechanism of membrane protein insertion into the endoplasmic reticulum (ER) has been extensively studied for many years (Shao and Hegde 2011). From this work, the signal recognition particle (SRP)/Sec61 pathway has emerged as a textbook example of a cotranslational membrane insertion mechanism (Grudnik et al. 2009). The SRP binds a hydrophobic segment (either a cleavable amino-terminal signal sequence or a transmembrane domain) immediately after it emerges from the ribosomal exit tunnel. This results in a translational pause that persists until SRP engages its receptor in the ER and delivers the ribosome-nascent chain complex to the Sec61 channel. Last, the Sec61 channel enables protein translocation into the ER lumen along with partitioning of hydrophobic transmembrane domains into the lipid bilayer through the Sec61 lateral gate (Rapoport 2007).Approximately 5% of all eukaryotic membrane proteins have an ER targeting signal in a single carboxy-terminal transmembrane domain that emerges from the ribosome exit tunnel following completion of protein synthesis and is not recognized by the SRP (Stefanovic and Hegde 2007). Nonetheless, because hydrophobic peptides in the cytoplasm are prone to aggregation and subject to degradation by quality control systems (Hessa et al. 2011), these tail-anchored (TA) proteins still have to be specifically recognized, shielded from the aqueous environment, and guided to the ER membrane for insertion. In the past five years, the guided-entry of TA proteins (GET) pathway has come to prominence as the major machinery for performing these tasks and the enabler of many key cellular processes mediated by TA proteins including vesicle fusion, membrane protein insertion, and apoptosis. This research has rapidly yielded biochemical and structural insights (​and2)2) into many of the GET pathway components (Hegde and Keenan 2011; Chartron et al. 2012a; Denic 2012). In particular, Get3 is an ATPase that uses metabolic energy to bridge recognition of TA proteins by upstream pathway components with TA protein recruitment to the ER for membrane insertion. However, the precise mechanisms of nucleotide-dependent TA protein binding to Get3 and how the GET pathway inserts tail anchors into the membrane are still poorly understood. Here, we provide an overview of the budding yeast GET pathway with emphasis on mechanistic insights that have come from structural studies of its membrane-associated steps and make a speculative juxtaposition with the ABC transporter mechanism.

Table 1.

A catalog of GET pathway component structures

	Pre-leptotene primary spermatocyte %	Pachytene primary spermatocyte %	Round spermatid %	Elongated spermatid %
DNA polymerase α	25	42	30	3
DNA polymerase β	29	34	36	1

Component	Role in the pathway	PDB ID
Sgt2	Component of the pretargeting complex that delivers TA proteins to Get3; dimer interacts with Get4/Get5, contains TPR repeats that interact with Hsps	3SZ7
Get5	Component of the pretargeting complex that delivers TA proteins to Get3; dimer interacts with Get4 via amino-terminal domain and with Sgt2 via its ubiquitin-like domain	2LNZ 3VEJ 2LO0
Get4	Component of the pretargeting complex that delivers TA proteins to Get3; interacts with Get3 via amino-terminal domain and with Get4 via carboxy-terminal domain	3LPZ 3LKU 3WPV
Get3	ATPase that binds the TA protein; dimer interacts with the pretargeting complex in the cytosol, and with Get1/2 at the ER membrane	Table 2
Get1	ER receptor for Get3; integral ER membrane protein, three TMDs; forms a complex with Get2	3SJA, 3SJB 3SJC, 3ZS8 3VLC, 3B2E
Get2	ER receptor for Get3; integral ER membrane protein, three TMDs; forms a complex with Get1	3SJD 3ZS9

Open in a separate windowTA, tail anchored; TPR, tetratricopeptide repeat; TMDs, transmembrane domains.

Table 2.

An itemized list of published Get3 structures with associated nucleotides and conformation nomenclature

Organism	Nucleotide	Conformation	PDB ID	References
Get3
Schizosaccharomyces pombe	None	Open	2WOO	Mateja et al. 2009
Saccharomyces cerevisiae	None	Open	3H84	Hu et al. 2009
			3A36	Yamagata et al. 2010
Aspergillus fumigatus	ADP	Open	3IBG	Suloway et al. 2009
S. cerevisiae	ADP	Open	3A37	Yamagata et al. 2010
Debaryomyces hansenii	ADP	Closed	3IO3	Hu et al. 2009
Chaetomium thermophilum	AMPPNP-Mg2+	Closed	3IQW	Bozkurt et al. 2009
C. thermophilum	ADP-Mg2+	Closed	3IQX	Bozkurt et al. 2009
S. cerevisiae	ADP•AlF4−-Mg2+	Fully closed	2WOJ	Mateja et al. 2009
Methanothermobacter thermautotrophicus	ADP•AlF4−-Mg2+	Fully closed	3ZQ6	Sherill et al. 2011
Methanococcus jannaschii	ADP•AlF4−-Mg2+	Tetrameric	3UG6	Suloway et al. 2012
			3UG7
Get3/Get2cyto
S. cerevisiae	ADP-Mg2+	Closed	3SJD	Stefer et al. 2011
S. cerevisiae	ADP•AlF4−-Mg2+	Closed	3ZS9	Mariappan et al. 2011
Get3/Get1cyto
S. cerevisiae	None	Semiopen	3SJC	Stefer et al. 2011
S. cerevisiae	ADP	Semiopen	3VLC	Kubota et al. 2012
S. cerevisiae	None	Open	3SJA	Stefer et al. 2011
			3SJB	Stefer et al. 2011
			3ZS8	Mariappan et al. 2011
	ADP	Open	3B2E	Kubota et al. 2012

Open in a separate windowADP, adenosine diphosphate.     

Endoplasmic Reticulum Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their carboxyl terminus. Many of these tail-anchored (TA) proteins are posttranslationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the guided-entry of TA protein insertion (GET) pathway. In recent years, most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway-targeting factor that uses nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pretargeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.The mechanism of membrane protein insertion into the endoplasmic reticulum (ER) has been extensively studied for many years (Shao and Hegde 2011). From this work, the signal recognition particle (SRP)/Sec61 pathway has emerged as a textbook example of a cotranslational membrane insertion mechanism (Grudnik et al. 2009). The SRP binds a hydrophobic segment (either a cleavable amino-terminal signal sequence or a transmembrane domain) immediately after it emerges from the ribosomal exit tunnel. This results in a translational pause that persists until SRP engages its receptor in the ER and delivers the ribosome-nascent chain complex to the Sec61 channel. Last, the Sec61 channel enables protein translocation into the ER lumen along with partitioning of hydrophobic transmembrane domains into the lipid bilayer through the Sec61 lateral gate (Rapoport 2007).Approximately 5% of all eukaryotic membrane proteins have an ER targeting signal in a single carboxy-terminal transmembrane domain that emerges from the ribosome exit tunnel following completion of protein synthesis and is not recognized by the SRP (Stefanovic and Hegde 2007). Nonetheless, because hydrophobic peptides in the cytoplasm are prone to aggregation and subject to degradation by quality control systems (Hessa et al. 2011), these tail-anchored (TA) proteins still have to be specifically recognized, shielded from the aqueous environment, and guided to the ER membrane for insertion. In the past five years, the guided-entry of TA proteins (GET) pathway has come to prominence as the major machinery for performing these tasks and the enabler of many key cellular processes mediated by TA proteins including vesicle fusion, membrane protein insertion, and apoptosis. This research has rapidly yielded biochemical and structural insights (​and2)2) into many of the GET pathway components (Hegde and Keenan 2011; Chartron et al. 2012a; Denic 2012). In particular, Get3 is an ATPase that uses metabolic energy to bridge recognition of TA proteins by upstream pathway components with TA protein recruitment to the ER for membrane insertion. However, the precise mechanisms of nucleotide-dependent TA protein binding to Get3 and how the GET pathway inserts tail anchors into the membrane are still poorly understood. Here, we provide an overview of the budding yeast GET pathway with emphasis on mechanistic insights that have come from structural studies of its membrane-associated steps and make a speculative juxtaposition with the ABC transporter mechanism.

Table 1.

A catalog of GET pathway component structures

Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

An experimental study using tissue culture medium as preservation and perfusion fluid

A commercially available tissue culture medium has been proven capable of preserving dog kidney function for at least 24 hr after simple cooling. The advantages of using tissue culture medium as preservation fluid instead of plasma or albumin solutions from the infectious and immunological points of view are obvious. An in vitro study was completed using the tissue

1.

     

15.

Diazomethane as a highly selective fatty acid methylating reagent for use in gas chromatographic analysis     

Hans W. Mueller 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,679(1-2)

     

16.

Beyond Specific Pathogen-Free: Biology and Effect of Common Viruses in Macaques     

Nicholas W Lerche  Joe H Simmons 《Comparative medicine》2008,58(1):8-10

     

17.

Ophthalmic artery chemosurgery for less advanced intraocular retinoblastoma: five year review     

Abramson DH  Marr BP  Brodie SE  Dunkel I  Palioura S  Gobin YP 《PloS one》2012,7(4):e34120

Background

Ophthalmic artery chemosurgery (OAC) for retinoblastoma was introduced by us 5 years ago for advanced intraocular retinoblastoma. Because the success was higher than with existing alternatives and systemic side effects limited we have now treated less advanced intraocular retinoblastoma (Reese-Ellsworth (RE) I-III and International Classification Retinoblastoma (ICRB) B and C).

Methodology/Principal Findings

Retrospective review of 5 year experience in eyes with Reese Ellsworth (

Code (animal No.)	Perfusion time (br)	Perfusion pressure mm/Hg	Flow ml/min	Weight gain	pH	pO2 mm/Hg	Histological appearance
1	24	70-60 systolic	96	35	7.3	150–180	Grossly normal
2	24		45-40 diastolic	108	30
3	24		96	30
4	48	70-60 systolic	80	35	7.3	150–180	Grossly normal
5	48	45-40 diastolic	120	40	7.4
6	48		100	40
7	72	70-60 systolic	115	40	7.4	150–180	Slight vacuolization of the tubular cells
8	72	45-40 diastolic	96	40
9	72		80	40
10	24	70-60 systolic	110	35	7.3	150–180	Used for transplantation
11	24	45-40 diastolic	120	35
12	24		140	40
13	24		100	30
14	24		96	30
Reese-Ellsworth (RE) Classification For Intraocular Retinoblastoma
GROUP I	a. Solitary tumor, less than 4 disc diameters in size, at or behind the equator
	b. Multiple tumors, none over 4 disc diameters in size, all at or behind the equator
GROUP II	a. Solitary tumor, less than 4 to 10 disc diameters in size, at or behind the equator
	b. Multiple tumors, none over 4 to 10 disc diameters in size, all at or behind the equator
GROUP III	a. Any lesion anterior to the equator
	b. Solitary tumors larger than 10 disc diameters behind the equator
GROUP IV	a. Multiple tumors, some larger than 10 disc diameters
	b. Any lesion extending anteriorly to the ora serrata
GROUP V	a . Massive tumors involving over half the retina
	b . Vitreous seeding

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Table 2

International Classification for Retinoblastoma (ICRB) Scheme.

International Classification for Intraocular Retinoblastoma (ICRB)
Group A	Small intraretinal tumors away from foveola and disc
	* All tumors are 3 mm or smaller in greatest dimension, confined to the retina and * All tumors are located further than 3 mm from the foveola and 1.5 mm from the optic disc
Group B	All remaining discrete tumors confined to the retina
	* All other tumors confined to the retina not in Group A * Tumor-associated subretinal fluid less than 3 mm from the tumor with no subretinal seeding
Group C	Discrete Local disease with minimal subretinal or vitreous seeding
	* Tumor(s) are discrete * Subretinal fluid, present or past, without seeding involving up to ¼ retina * Local fine vitreous seeding may be present close to discrete tumor * Local subretinal seeding less than 3 mm (2DD) from the tumor
Group D	Diffuse disease with significant vitreous or subretinal seeding
	* Tumor(s) may be massive or diffuse * Subretinal fluid, present or past without seeding, involving up to total retinal detachment * Diffuse or massive vitreous disease may include “greasy” seeds or avascular tumor masses * Diffuse subretinal seeding may include subretinal plaques or tumor nodules
Group E	Presence of any one or more of these poor prognosis features
	* Tumor touching the lens * Tumor anterior to anterior vitreous face involving ciliary body or anterior segment * Diffuse infiltrating retinoblastoma * Neovascular glaucoma * Opaque media from hemorrhage * Tumor necrosis with aseptic orbital cellulites * Phthisis bulbi

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Conclusions/Significance

Ophthalmic artery chemosurgery for retinoblastoma that was Reese-Ellsworth I, II and III (or International Classification B or C) was associated with high success (100% of treatable eyes were retained) and limited toxicity with results that equal or exceed conventional therapy with less toxicity.     

Ophthalmic artery chemosurgery for less advanced intraocular retinoblastoma: five year review

Background

Ophthalmic artery chemosurgery (OAC) for retinoblastoma was introduced by us 5 years ago for advanced intraocular retinoblastoma. Because the success was higher than with existing alternatives and systemic side effects limited we have now treated less advanced intraocular retinoblastoma (Reese-Ellsworth (RE) I-III and International Classification Retinoblastoma (ICRB) B and C).

Methodology/Principal Findings

Retrospective review of 5 year experience in eyes with Reese Ellsworth (

Integrity of lysosomes in the isolated perfused rat liver before and after exposure to dimethylsulphoxide.

We have studied the integrity of lysosomes in isolated rat livers perfused for 3, 4, or 6 hr at 35 °C with BSA (40 g/l) in Krebs Ringer bicarbonate buffer. The latency and sedimentability of β-glucuronidase in homogenates of these livers was well maintained even after 6 hr. The latency and sedimentability of acid phosphatase remained at about control levels during the first 4 hr of perfusion but decreased between 4 and 6 hr. These decreases in latency and sedimentability correlated with a decrease in bile production and an increase in the rate of release of GOT into the perfusate and could indicate either intracellular disruption of lysosomes

Latencies, Sedimentabilities, and Specific Activities of Acid Phosphatase and β-Glucuronidase in Homogenates of Rat Liver Prepared before or at Various Times after Exposure to 1.4 m Me₂SO for 1 hr

     

20.

Heat exchange in the cryosurgery of Menière's disease: Experimental and clinical studies     

S. Harly  J.E. Aastrup  O. Elbrønd 《Cryobiology》1977,14(5):609-613

The temperature course in the lateral semicircular canal and in the facial canal was studied in experiments during freezing of the semicircular canal. The course of the temperature was measured with thermocouples. Concurrently, the heat flow was measured, and also the total heat exchange was measured throughout the freezing period by a thermoelectric heat flowmeter incorporated in the cryotip. The measurements showed correlation between the total amount of heat exchanged, the freezing time, and the temperature in the semicircular canal. This correlation was utilized to assess and calculate (the temperature of the lateral semicircular canal) the course of the cryoprocess in vivo, where it is possible to measure the heat flow and the total heat exchange during the freezing period only.

2. Results upon Vertigo

Time (hr)	0	3	4	6
Acid phosphatase
Latency (%)	83.2 ± 0.8	64.7 ± 5.1	62.4 ± 7.9	68.9 ± 5.4
Sedimentability (%)	81.7 ± 0.6	77.6 ± 3.1	81.0 ± 3.4	79.4 ± 5.1
Specific activity (mIU/mg protein)	2.8 ± 0.4	2.8 ± 0.2	2.4 ± 0.4	1.6 ± 0.2
β-Glucuronidase
Latency (%)	06.8 ± 1.5	71.3 ± 4.3	74.2 ± 2.5	63.3 ± 4.5
Sedimentability (%)	69.5 ± 0.3	75.4 ± 3.2	75.0 ± 1.1	74.6 ± 2.0
Specific activity (mIU/mg protein)	1.8 ± 0.1	1.6 ± 0.1	1.6 ± 0.2	1.6 ± 0.2

     

	No Vertigo	Improved	Unchanged
Number of patients	7	5	3

Heat exchange in the cryosurgery of Menière's disease: Experimental and clinical studies

The temperature course in the lateral semicircular canal and in the facial canal was studied in experiments during freezing of the semicircular canal. The course of the temperature was measured with thermocouples. Concurrently, the heat flow was measured, and also the total heat exchange was measured throughout the freezing period by a thermoelectric heat flowmeter incorporated in the cryotip. The measurements showed correlation between the total amount of heat exchanged, the freezing time, and the temperature in the semicircular canal. This correlation was utilized to assess and calculate (the temperature of the lateral semicircular canal) the course of the cryoprocess in vivo, where it is possible to measure the heat flow and the total heat exchange during the freezing period only.

2. Results upon Vertigo