Insoluble Pool Research Articles

Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids)

BackgroundThe importance of uridine 5′-diphosphate glucose (UDP-G) synthesis and degradation on carbon (C) partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of involved enzymes had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools. In this study, the expression of 136 genes belonging to 32 gene families related to UDP-G metabolism was studied in 3 major sugarcane organs (including leaf, internode and root) at 6 different developmental stages in 2 commercial genotypes.ResultsAnalysis of the genes associated with UDP-G metabolism in leaves indicated low expression of sucrose synthase, but relatively high expression of invertase genes, specifically cell-wall invertase 4 and neutral acid invertase 1–1 and 3 genes. Further, organs that are primarily responsible for sucrose synthesis or bioaccumulation, i.e., in source organs (mature leaves) and storage sink organs (mature internodes), had very low expression of sucrose, cellulose and hemicellulose synthesis genes, specifically sucrose synthase 1 and 2, UDP-G dehydrogenase 5 and several cellulose synthase subunit genes. Gene expression was mostly very low in both leaf and mature internode samples; however, leaves did have a comparatively heightened invertase and sucrose phosphate synthase expression. Major differences were observed in the transcription of several genes between immature sink organs (roots and immature internodes). Gene transcription favoured utilisation of UDP-G toward insoluble and respiratory pools in roots. Whereas, there was comparatively higher expression of sucrose synthetic genes, sucrose phosphate synthase 1 and 4, and comparatively lower expression of many genes associated with C flow to insoluble and respiratory pools including myo-Inositol oxygenase, UDP-G dehydrogenase 4, vacuolar invertase 1, and several cell-wall invertases in immature internodes.ConclusionThis study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in sugarcane. Transcriptional analysis displayed the likelihood that C partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism. The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

Bioactivation of Legacy Phosphorus in Calcareous Soil and its Impact on Maize Yield

Background: Over the years, the excessive P fertilizer application to crop fields resulted in the build up of insoluble phosphorus pool in soil known as legacy phosphorus. This represents a vast and largely untapped resource in soil. The current study was conducted to evaluate the potential of some P activators (Phosphorus solubilising bacteria, phytase, humic acid, farmyard manure) on increasing the plant availability of legacy P in calcareous soil. Methods: A field experiment was conducted with maize hybrid COH(M) 6. The P activators were combined and applied along with the different doses of P fertilizer (100%, 75% and 50% soil test dose of single super phosphate). Result: Farmyard Manure (FYM) application with Humic acid (HA) showed the increased Olsen P under 100% and 75% of P application. The phosphorus activation response of FYM+HA applied plots showed a higher value than other treatments. The maize yield and P uptake obtained with FYM and HA were statistically comparable at 100% and 75% soil test dose of P. The findings have illustrated that even the reduced amount of P fertilizer application can support the crop growth due to the release of legacy P if they are applied along with P- activators such as FYM and HA.

Citrullination of Amyloid-β Peptides in Alzheimer's Disease.

Protein citrullination (deimination of arginine residue) is a well-known biomarker of inflammation. Elevated protein citrullination has been shown to colocalize with extracellular amyloid plaques in postmortem AD patient brains. Amyloid-β (Aβ) peptides which aggregate and accumulate in the plaques of Alzheimer's disease (AD) have sequential N-terminal truncations and multiple post-translational modifications (PTM) such as isomerization, pyroglutamate formation, phosphorylation, nitration, and dityrosine cross-linking. However, no conclusive biochemical evidence exists whether citrullinated Aβ is present in AD brains. In this study, using high-resolution mass spectrometry, we have identified citrullination of Aβ in sporadic and familial AD brains by characterizing the tandem mass spectra of endogenous N-truncated citrullinated Aβ peptides. Our quantitative estimations demonstrate that ∼ 35% of pyroglutamate3-Aβ pool was citrullinated in plaques in the sporadic AD temporal cortex and ∼ 22% in the detergent-insoluble frontal cortex fractions. Similarly, hypercitrullinated pyroglutamate3-Aβ (∼ 30%) was observed in both the detergent-soluble as well as insoluble Aβ pool in familial AD cases. Our results indicate that a common mechanism for citrullination of Aβ exists in both the sporadic and familial AD. We establish that citrullination of Aβ is a remarkably common PTM, closely associated with pyroglutamate3-Aβ formation and its accumulation in AD. This may have implications for Aβ toxicity, autoantigenicity of Aβ, and may be relevant for the design of diagnostic assays and therapeutic targeting.

G protein-coupled receptor kinases are associated with Alzheimer's disease pathology.

Alzheimer's disease (AD) is characterised by extracellular deposition of amyloid-β (Aβ) in amyloid plaques and intracellular aggregation and accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs). Although several kinases have been identified to contribute to the pathological phosphorylation of tau, kinase-targeted therapies for AD have not been successful in clinical trials. Critically, the kinases responsible for numerous identified tau phosphorylation sites remain unknown. G protein-coupled receptor (GPCR) kinases (GRKs) have recently been implicated in phosphorylation of non-GPCR substrates, for example, tubulin and α-synuclein, and in neurological disorders, including schizophrenia and Parkinson's disease. Accordingly, we investigated the involvement of GRKs in the pathophysiology of AD. We performed a comprehensive immunohistochemical and biochemical analysis of the ubiquitously expressed GRKs, namely, GRK2, 3, 5 and 6, in postmortem human brain tissue of control subjects and AD patients. GRKs display unique cell-type-specific expression patterns in neurons, astrocytes and microglia. Levels of GRKs 2, 5 and 6 are specifically decreased in the CA1 region of the AD hippocampus. Biochemical evidence indicates that the GRKs differentially associate with total, soluble and insoluble pools of tau in the AD brain. Complementary immunohistochemical studies indicate that the GRKs differentially colocalise with total tau, phosphorylated tau and NFTs. Notably, GRKs 3 and 5 also colocalise with amyloid plaques. These studies establish a link between GRKs and the pathological phosphorylation and accumulation of tau and amyloid pathology in AD brains and suggest a novel role for these kinases in regulation of the pathological hallmarks of AD.

Interaction of the Anti-Proliferative GPER Inverse Agonist ERα17p with the Breast Cancer Cell Plasma Membrane: From Biophysics to Biology.

The peptide ERα17p, which corresponds to the 295-311 fragment of the hinge/AF2 domains of the human estrogen receptor α (ERα), exerts apoptosis in breast cancer cells through a mechanism involving the G protein-coupled estrogen-dependent receptor GPER. Besides this receptor-mediated mechanism, we have detected a direct interaction (Kd value in the micromolar range) of this peptide with lipid vesicles mimicking the plasma membrane of eukaryotes. The reversible and not reversible pools of interacting peptide may correspond to soluble and aggregated membrane-interacting peptide populations, respectively. By using circular dichroism (CD) spectroscopy, we have shown that the interaction of the peptide with this membrane model was associated with its folding into β sheet. A slight leakage of the 5(6)-fluorescein was also observed, indicating lipid bilayer permeability. When the peptide was incubated with living breast cancer cells at the active concentration of 10 μM, aggregates were detected at the plasma membrane under the form of spheres. This insoluble pool of peptide, which seems to result from a fibrillation process, is internalized in micrometric vacuoles under the form of fibrils, without evidence of cytotoxicity, at least at the microscopic level. This study provides new information on the interaction of ERα17p with breast cancer cell membranes as well as on its mechanism of action, with respect to direct membrane effects.

TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole

Amyotrophic lateral sclerosis (ALS) represents a fatal neurodegenerative disease, which is characterized by a rapid loss of lower and upper motor neurons. As a major neuropathological hallmark, protein aggregates containing the Transactivating Response Region (TAR) DNA Binding Protein (TDP-43) are detectable in about 95% of sporadic ALS patients. TDP-43 interacts with itself physiologically to form liquid droplets, which may progress to pathological aggregates. In this study, we established the NanoBit luciferase complementation assay to measure TDP-43 self-interaction and found the fusion of the split luciferase subunits to the N-terminus of the protein as the strongest interacting partners. A screen of pharmacologically active compounds from the LOPAC®1280 library identified auranofin, chelerythrine and riluzole as dose-dependent inhibitors of TDP-43 self-interaction. Further analysis of drug action of the gold-containing thioredoxin reductase inhibitor auranofin revealed a redistribution from insoluble TDP-43 protein pool to PBS-soluble protein pool in N2a cells. In addition, auranofin treatment diminished reduced glutathione as a sign for oxidative modulation.

Costs of acquiring phosphorus by vascular land plants: patterns and implications for plant coexistence.

Content Summary 1420 I. Introduction 1421 II. Root adaptations that influence P acquisition 1422 III. Costs of P acquisition: general 1423 IV. Costs of P acquisition that are independent of soil P concentrations 1423 V. Costs of P acquisition that increase as soil P concentrations decline 1424 VI. Discussion and conclusions 1424 Acknowledgements 1425 References 1425 SUMMARY: We compare carbon (and hence energy) costs of the different modes of phosphorus (P) acquisition by vascular land plants. Phosphorus-acquisition modes are considered to be mechanisms of plants together with their root symbionts and structures such as cluster roots involved in mobilising or absorbing P. Phosphorus sources considered are soluble and insoluble inorganic and organic pools. Costs include operating the P-acquisition mechanisms, and resource requirements to construct and maintain them. For most modes, costs increase as the relevant soil P concentration declines. Costs can thus be divided into a component incurred irrespective of soil P concentration, and a component describing how quickly costs increase as the soil P concentration declines. Differences in sensitivity of costs to soil P concentration arise mainly from how economically mycorrhizal fungal hyphae or roots that explore the soil volume are constructed, and from costs of exudates that hydrolyse or mobilise insoluble P forms. In general, modes of acquisition requiring least carbon at high soil P concentrations experience a steeper increase in costs as soil P concentrations decline. The relationships between costs and concentrations suggest some reasons why different modes coexist, and why the mixture of acquisition modes differs between sites.

P.144 - Turnover studies on DNAJB6 and the CASA pathway proteins

Limb-girdle muscular dystrophy type 1D (LGMD1D) results from dominant mutations in the co-chaperone DNAJB6. The mutations confer toxic properties to the short cytoplasmic isoform DNAJB6b, and slow down the turnover of mutant DNAJB6, as well as that of the co-expressed wild-type protein. We have previously shown that DNAJB6 interacts with the chaperone-assisted selective autophagy (CASA) system. This macroautophagy pathway, important for muscle maintenance, depends on the co-chaperone BAG3, together with HSPA8 (Hsc70) and HSPB8 (Hsp22). Interestingly, overexpression of BAG3 increases DNAJB6b toxicity in zebrafish – an effect not seen with the BAG3 myopathy mutant P209L – suggesting an active role for BAG3 in LGMD1D pathogenesis. The role of altered DNAJB6 turnover in the pathogenesis of LGMD1D, and its relationship with the CASA proteins is unknown. BAG3 could increase the toxicity of mutant DNAJB6b by aggravating its turnover block. Alternatively, if BAG3 and HSPB8 lie downstream of DNAJB6b mutations, also their turnover rates could be affected. To explore these possibilities, we have carried out cell-culture based protein turnover studies. In our setup, proteins of interest are expressed in a tetracycline-inducible system, and their turnover is followed after tetracycline removal. Coexpression or knockdown allows identification of modulating effects of other proteins. Unexpectedly, while wild-type BAG3 had little effect, BAG3 P209L blocked the turnover of both wild-type and mutant DNAJB6b. This correlated with a shift of DNAJB6b to the insoluble fraction, suggesting that mutant BAG3 may sequester DNAJB6b into an insoluble pool that is resistant to normal degradation. Our results reinforce a functional interaction between DNAJB6 and BAG3, provide a possible explanation for the different effects of wild-type and P209L BAG3 on DNAJB6 toxicity, and place DNAJB6 downstream of the BAG3 P209L mutation, potentially linking the pathomechanisms of LGMD1D and BAG3 myopathy.

Patterns and regulation of silicon accumulation in Synechococcus spp.

Six clones of the marine cyanobacterium Synechococcus, representing four major clades, were all found to contain significant amounts of silicon in culture. Growth rate was unaffected by silicic acid, Si(OH)4 , concentration between 1 and 120μM suggesting that Synechococcus lacks an obligate need for silicon (Si). Strains contained two major pools of Si: an aqueous soluble and an aqueous insoluble pool. Soluble pool sizes correspond to estimated intracellular dissolved Si concentrations of 2-24mM, which would be thermodynamically unstable implying the binding of intracellular soluble Si to organic ligands. The Si content of all clones was inversely related to growth rate and increased with higher [Si(OH)4 ] in the growth medium. Accumulation rates showed a unique bilinear response to increasing [Si(OH)4 ] from 1 to 500μM with the rate of Si acquisition increasing abruptly between 80 and 100μM Si(OH)4 . Although these linear responses imply some form of diffusion-mediated transport, Si uptake rates at low Si (~1μM Si) were inhibited by orthophosphate, suggesting a role of phosphate transporters in Si acquisition. Theoretical calculations imply that observed Si acquisition rates are too rapid to be supported by lipid-solubility diffusion of Si through the plasmalemma; however, facilitated diffusion involving membrane protein channels may suffice. The data are used to construct a working model of the mechanisms governing the Si content and rate of Si acquisition in Synechococcus.

Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown. Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and high pressure liquid chomatography with electrochemical detection analysis. We found that APAP-AD were detected at 1h, peaked at approximately 2h, declined at 6h and almost full removed at 24h post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were colocalized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes. Our data indicate that APAP-AD are removed though selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury. Acetaminophen overdose can form acetaminophen protein adducts and mitochondria damage in hepatocytes resulting in liver injury. Activation of autophagy-lysosomal degradation pathway can help to remove acetaminophen protein adducts. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

Insoluble prokaryotic membrane lipids in a Sphagnum peat: Implications for organic matter preservation

Preservation of organic matter (OM) in the geosphere has a direct impact on carbon bioavailability, the carbon cycle and the formation of fossil fuels. We have examined some of the processes that lead to the preservation of OM by characterising insoluble OM in a Sphagnum peat bog. We focussed on the partitioning of prokaryotic biomarkers between solvent-extractable and insoluble OM fractions and how that partitioning changed with depth. The insoluble organic matter (IOM) was examined using stepwise chemical degradation involving base and acid hydrolysis. Iso- and anteiso-C15 and C17 fatty acids (FAs), hopanoic acid and bishomohopanol, and branched glycerol dialkyl glycerol tetraethers (GDGTs) – diagnostic for Bacteria – were targeted as well as archaeol and isoprenoidal GDGTs – diagnostic for Archaea. High percentages of these compounds – up to 65% – occur in IOM pools, indicating that archaeal- and bacterially derived OM is prone to insolubilization even in shallow sediments (<5cm depth). Differences in functionalities – likely related to intact polar (IP) head groups – seem to determine the insolubilization of prokaryotic lipids; specifically, we propose that compounds bearing rapidly degradable phosphate-based polar head groups are less likely to be incorporated into the insoluble OM pools. Collectively, these data indicate that microbial membrane lipids are rapidly incorporated into peat IOM during early diagenesis. We suggest that this is due to an inherently recalcitrant character of some prokaryotic cells which affords protection to their membrane lipids. However, this large proportion of insoluble prokaryotic lipids does not appear to be entirely stable at shallow burial depths, indicating that the peat IOM is a dynamic reservoir, at least during early diagenesis.

Non-structural carbon dynamics and allocation relate to growth rate and leaf habit in California oaks.

Trees contain non-structural carbon (NSC), but it is unclear for how long these reserves are stored and to what degree they are used to support plant activity. We used radiocarbon ((14)C) to show that the carbon (C) in stemwood NSC can achieve ages of several decades in California oaks. We separated NSC into two fractions: soluble (∼50% sugars) and insoluble (mostly starch) NSC. Soluble NSC contained more C than insoluble NSC, but we found no consistent trend in the amount of either pool with depth in the stem. There was no systematic difference in C age between the two fractions, although ages increased with stem depth. The C in both NSC fractions was consistently younger than the structural C from which they were extracted. Together, these results indicate considerable inward mixing of NSC within the stem and rapid exchange between soluble and insoluble pools, compared with the timescale of inward mixing. We observed similar patterns in sympatric evergreen and deciduous oaks and the largest differences among tree stems with different growth rates. The (14)C signature of carbon dioxide (CO2) emitted from tree stems was higher than expected from very recent photoassimilates, indicating that the mean age of C in respiration substrates included a contribution from C fixed years previously. A simple model that tracks NSC produced each year, followed by loss (through conversion to CO2) in subsequent years, matches our observations of inward mixing of NSC in the stem and higher (14)C signature of stem CO2 efflux. Together, these data support the idea of continuous accumulation of NSC in stemwood and that 'vigor' (growth rate) and leaf habit (deciduous vs evergreen) control NSC pool size and allocation.

Cortical pyroglutamate amyloid-β levels and cognitive decline in Alzheimer's disease

Posterior cingulate cortex (PCC) accumulates amyloid-β (Aβ) early in Alzheimer's disease (AD). The relative concentrations of full-length Aβ and truncated, pyroglutamate-modified Aβ (NpE3) forms, and their correlations to cognitive dysfunction in AD, are unknown. We quantified AβNpE3-42, AβNpE3-40, Aβ1-42, and Aβ1-40 concentrations in soluble (nonfibrillar) and insoluble (fibrillar) pools in PCC from subjects with an antemortem clinical diagnosis of no cognitive impairment, mild cognitive impairment, or mild-moderate AD. In clinical AD, increased PCC concentrations of Aβ were observed for all Aβ forms in the insoluble pool but only for Aβ1-42 in the soluble pool. Lower Mini-Mental State Exam and episodic memory scores correlated most strongly with higher concentrations of soluble and insoluble Aβ1-42. Greater neuropathology severity by Consortium to Establish a Registry for Alzheimer's Disease and National Institute on Aging-Reagan pathologic criteria was associated with higher concentrations of all measured Aβ forms, except soluble AβNpE3-40. Low concentrations of soluble pyroglutamate Aβ across clinical groups likely reflect its rapid sequestration into plaques, thus, the conversion to fibrillar Aβ may be a therapeutic target.

Pathways for Neoarchean pyrite formation constrained by mass-independent sulfur isotopes.

It is generally thought that the sulfate reduction metabolism is ancient and would have been established well before the Neoarchean. It is puzzling, therefore, that the sulfur isotope record of the Neoarchean is characterized by a signal of atmospheric mass-independent chemistry rather than a strong overprint by sulfate reducers. Here, we present a study of the four sulfur isotopes obtained using secondary ion MS that seeks to reconcile a number of features seen in the Neoarchean sulfur isotope record. We suggest that Neoarchean ocean basins had two coexisting, significantly sized sulfur pools and that the pathways forming pyrite precursors played an important role in establishing how the isotopic characteristics of each of these pools was transferred to the sedimentary rock record. One of these pools is suggested to be a soluble (sulfate) pool, and the other pool (atmospherically derived elemental sulfur) is suggested to be largely insoluble and unreactive until it reacts with hydrogen sulfide. We suggest that the relative contributions of these pools to the formation of pyrite depend on both the accumulation of the insoluble pool and the rate of sulfide production in the pyrite-forming environments. We also suggest that the existence of a significant nonsulfate pool of reactive sulfur has masked isotopic evidence for the widespread activity of sulfate reducers in the rock record.

Microbial N immobilization is of great importance in acidified mountain spruce forest soils

The prevailing N saturation paradigm still considers microbial N immobilization as a less important process of ecosystem N retention. On the contrary, we hypothesize that it can even be a primary process affecting N leaching from N saturated soils. We studied N transformations in soils of acidified near-natural and primeval forests in the Bohemian Forest (watersheds of Plešné and Čertovo Lakes, Czech Republic) and Pop Ivan Massif (Ukraine). Organic soils were sampled from similar conditions (1100–1500 m a.s.l., precipitation 1400–1800 mm, acidic bedrock, forest dominated by Picea abies) and had similar chemical properties (pHKCl 2.5–3.2, BS ∼45%, Alex ∼40 meq kg−1). However, the Ukrainian soil had lower soil C/N ratio (24 vs. 30) and C availability (water soluble C and C/N ratio of 65 vs. ∼114–163 μmol g−1 and 6 vs. 21–24, respectively) than the other soils. We ran laboratory experiments in which mixtures of different N sources (N–NH4, N–NO3 and glycine) were added to the soil with only one source 15N-labelled. We followed 15N partitioning within soil N pools and analysed the composition of the microbial community (16SrDNA-DGGE fingerprint of bacteria, ergosterol analyses, qPCR of fungal 18S rDNA gene). The microbial N pool was always three to five times higher than the total soluble N pool. We found rapid (15 min) and simultaneous immobilization of all added N forms into the microbial biomass with clear preferences for organic N over inorganic sources. The total N flux to the microbial pool always exceeded N flux into mineral N pools. The pattern of N transformation in the C limited soil was different from the other soils. The microbial pool and N flux into it were smaller compared to the mineral N pools and fluxes. The contribution of N–NO3 to microbial immobilization was negligible, while nitrification was almost equal to N mineralization. Total N flux through soluble N pools was greater than total N flux to insoluble pools (residual and microbial N); this was accompanied by lower microbial N uptake efficiency and shorter residence time of N in microbial pool than in soils with higher C availability. The composition of bacterial community was related to DOC content and C and N in microbial biomass. In soils with higher fungal abundance, more glycine was immobilized regardless of soil C availability, but with higher deamination (∼50 vs. 20%) and subsequent release of N–NH4 back to the soil. Our study emphasized the role of microbial N immobilization in preventing N–NO3 loss from N saturated ecosystems as a function of C availability. Nitrification was favoured when enough N–NH4 was available in the C limited soil. The discharged N–NO3 was not immobilized by the microbes and could be, if not immobilized by plants, leached out. C limitation plays an important role in the susceptibility of ecosystems to N leaching and could partially explain the observed differences in some N-saturated ecosystems.

Effect of Variable Sugar Concentrations on Glycogen and Ethanol Content in Saccharomyces sp.

Saccharomyces cerevisiae (MTCC-12) maintains two pools of glycogen, one soluble and the other cell wall bound insoluble glycogen. Effect of variable concentrations of sugar on the insoluble pool of glycogen linked to wall β-glucan component was found to be more significant than that observed for soluble pool of glycogen. The study revealed that cells exhibit exponential increase in the amount of insoluble glycogen with corresponding increase in sugar concentration from 2-10 % (w/v). Ethanol content as well as concentration of insoluble glycogen in yeast cells, which were grown in sucrose medium, shows higher values than in glucose medium. Parallel increase in glycogen and ethanol content indicates their correlation with each other. Exponential increase in insoluble glycogen content serves as a protective measure against ethanol stress created across the plasma membrane of yeast cell.

Myosin Va Is Required for P Body but Not Stress Granule Formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.

Exercise training does not correct abnormal cardiac glycogen accumulation in thedb/dbmouse model of type 2 diabetes

Substrate imbalance is a well-recognized feature of diabetic cardiomyopathy. Insulin resistance effectively limits carbohydrate oxidation, resulting in abnormal cardiac glycogen accumulation. Aims of the present study were to 1) characterize the role of glycogen-associated proteins involved in excessive glycogen accumulation in type 2 diabetic hearts and 2) determine if exercise training can attenuate abnormal cardiac glycogen accumulation. Control (db(+)) and genetically diabetic (db/db) C57BL/KsJ-lepr(db)/lepr(db) mice were subjected to sedentary or treadmill exercise regimens. Exercise training consisted of high-intensity/short-duration (10 days) and low-intensity/long-duration (6 wk) protocols. Glycogen levels were elevated by 35-50% in db/db hearts. Exercise training further increased (2- to 3-fold) glycogen levels in db/db hearts. Analysis of soluble and insoluble glycogen pools revealed no differential accumulation of one glycogen subspecies. Phosphorylation (Ser(640)) of glycogen synthase, an indicator of enzymatic fractional activity, was greater in db/db mice subjected to sedentary and exercise regimens. Elevated glycogen levels were accompanied by decreased phosphorylation (Thr(172)) of 5'-AMP-activated kinase and phosphorylation (Ser(79)) of its downstream substrate acetyl-CoA carboxylase. Glycogen concentration was not associated with increases in other glycogen-associated proteins, including malin and laforin. Novel observations show that exercise training does not correct diabetes-induced elevations in cardiac glycogen but, rather, precipitates further accumulation.

Desmosome Disassembly in Response to Pemphigus Vulgaris IgG Occurs in Distinct Phases and Can Be Reversed by Expression of Exogenous Dsg3

Pemphigus vulgaris (PV) is an epidermal blistering disorder caused by antibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). The mechanism by which PV IgG disrupts adhesion is not fully understood. To address this issue, primary human keratinocytes (KCs) and patient IgG were used to define the morphological, biochemical, and functional changes triggered by PV IgG. Three phases of desmosome disassembly were distinguished. Analysis of fixed and living KCs demonstrated that PV IgG cause rapid Dsg3 internalization, which likely originates from a non-junctional pool of Dsg3. Subsequently, Dsg3 and other desmosomal components rearrange into linear arrays that run perpendicular to cell contacts. Dsg3 complexes localized at the cell surface are transported in a retrograde manner along with these arrays before being released into cytoplasmic vesicular compartments. These changes in Dsg3 distribution are followed by depletion of detergent-insoluble Dsg3 pools and by the loss of cell adhesion strength. Importantly, this process of disassembly can be prevented by expressing exogenous Dsg3, thereby driving Dsg3 biosynthesis and desmosome assembly. These data support a model in which PV IgG cause the loss of cell adhesion by altering the dynamics of Dsg3 assembly into desmosomes and the turnover of cell surface pools of Dsg3 through endocytic pathways.

Role of Insoluble Glycogen in Ethanol Adaptation Mechanism of Saccharomyces italicus

Saccharomyces italicus has two pools of glycogen-one is the soluble pool and the remainder water insoluble linked with cell wall or β-glucans. Soluble pool of glycogen present is assigned to produce energy required for metabolic activities but the insoluble pool in the cell wall plays a special physiological role in providing the osmotolerance under ethanol stress conditions. S. italicus grown in the presence of 2-8% v/v ethanol exhibits exponential increase in insoluble glycogen content as a protective measure against the stress created across the plasma membrane by the action of ethanol. But ethanol concentration above 8%v/v reduces hydrophobic interactions thereby decreasing the insoluble glycogen content with increase in membrane permeability.