Low Molecular Weight Heat Shock Research Articles

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-β1 Signaling.

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Underpinning the molecular programming attributing heat stress associated thermotolerance in tea (Camellia sinensis (L.) O. Kuntze)

The most daunting issue of global climate change is the deleterious impact of extreme temperatures on tea productivity and quality, which has resulted in a quest among researchers and growers. The current study aims to unravel molecular programming underpinning thermotolerance by characterizing heat tolerance and sensitivity response in 20 tea cultivars. The significantly higher negative influence of heat stress was recorded in a sensitive cultivar with reduced water retention (47%), chlorophyll content (33.79%), oxidation potential (32.48%), and increase in membrane damage (76.4%). Transcriptional profiling of most tolerant and sensitive cultivars identified 78 differentially expressed unigenes with chaperon domains, including low and high molecular weight heat shock protein (HSP) and heat shock transcription factors (HSFs) involved in heat shock response (HSR). Further, predicted transcriptional interactome network revealed their key role in thermotolerance via well-co-ordinated transcriptional regulation of aquaporins, starch metabolism, chlorophyll biosynthesis, calcium, and ethylene mediated plant signaling system. The study identified the key role of HSPs (CsHSP90) in regulating HSR in tea, wherein, structure-based molecular docking revealed the inhibitory role of geldanamycin (GDA) on CsHSP90 by blocking ATP binding site at N-terminal domain of predicted structure. Subsequently, GDA mediated leaf disc inhibitor assay further affirmed enhanced HSR with higher expression of CsHSP17.6, CsHSP70, HSP101, and CsHSFA2 genes in tea. Through the current study, efforts were made to extrapolate a deeper understanding of chaperons mediated regulation of HSR attributing thermotolerance in tea.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

Construction and analysis of an Oryza sativa (cv. MR219) salinity-related cDNA library

To identify salt-responsive genes, we constructed a cDNA subtractive library from a salt-tolerant rice cultivar, MR219. Treatments of the whole plant were carried out with 150 mM sodium chloride for 0, 2, 4, 8, 12 and 24 h. The extraction of total RNA from 14-day-old seedlings was conducted on the shoots and roots of both treated and untreated plants. The treated plants were utilized as ‘drivers’ while the untreated plants were utilized as ‘testers’. Equal amounts of the extracted total RNA from the 12 samples were pooled together and subjected to mRNA isolation. A reverse transcription of tester and driver cDNAs from the mRNA of two pools of the samples was conducted. In total, 300 clones were sequenced and analysed using bioinformatics tools. The 260 high-quality sequences were assembled and 83 contigs and 42 singletons were generated, producing a total of 125 UTs. The majority of the UTs showed significant homology with sequences in the NCBI database. A total of 330 gene ontology terms were grouped into three categories, of which 144 UTs fell under biological process, 108 fell under cellular component and 78 fell under molecular function. Also, RT-PCR was utilized to substantiate the elevation in expression of six candidate genes ((MR219SAP8 (JZ532324) and salT (JZ532363), low molecular weight heat shock (JZ532334), phospholipid hydroperoxide glutathione (JZ532371), Acetamidase (JZ532408), Ferredoxin-1 and chloroplastic (JZ532374)) subjected to salinity stress at varying timepoints.

An RNA sequencing transcriptome analysis of the high-temperature stressed tall fescue reveals novel insights into plant thermotolerance.

BackgroundTall fescue (Festuca arundinacea Schreb.) is major cool-season forage and turf grass species worldwide, but high-temperature is a major environmental stress that dramatically threaten forage production and turf management of tall fescue. However, very little is known about the whole-genome molecular mechanisms contributing to thermotolerance. The objectives of this study were to analyzed genome-wide gene expression profiles in the leaves of two tall fescue genotypes, heat tolerant ‘PI578718’ and heat sensitive ‘PI234881’ using high-throughput RNA sequencing.ResultsA total of 262 million high-quality paired-end reads were generated and assembled into 31,803 unigenes with an average length of 1,840 bp. Of these, 12,974 unigenes showed different expression patterns in response to heat stress and were categorized into 49 Gene Ontology functional subcategories. In addition, the variance of enrichment degree in each functional subcategory between PI578718 and PI234881 increased with increasing treatment time. Cell division and cell cycle genes showed a massive increase in transcript abundance in heat-stressed plants and more activated genes were detected in PI 578718 by Kyoto Encyclopedia of Genes and Genomes pathways analysis. Low molecular weight heat shock protein (LMW-HSP, HSP20) showed activated in two stressed genotypes and high molecular weight HSP (HMW-HSP, HSP90) just in PI578718. Assimilation such as photosynthesis, carbon fixation, CH4, N, S metabolism decreased along with increased dissimilation such as oxidative phosphorylation.ConclusionsThe assembled transcriptome of tall fescue could serve as a global description of expressed genes and provide more molecular resources for future functional characterization analysis of genomics in cool-season turfgrass in response to high-temperature. Increased cell division, LMW/HMW-HSP, dissimilation and antioxidant transcript amounts in tall fescue were correlated with successful resistance to high temperature stress.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1147) contains supplementary material, which is available to authorized users.

MANAGING CHILLING INJURY IN VEGETABLES

Common vegetables that are susceptible to chilling injury include asparagus, beans, cucumbers, eggplants, ginger, jicama, okra, peppers, potatoes, pumpkins, sweet potatoes, tamarillos, tomatoes, and zucchini squashes. Most of the susceptible crops are either fruit vegetables or root vegetables. Chilling injury symptoms vary depending on the commodity, but generally manifest as pitting, discoloration, internal breakdown, and decay. Images from nuclear magnetic resonance revealed that signals on the peripheral of cells in chilled zucchini tissues were much more intense than those in non-chilled tissues, indicating that cell membranes were damaged by chilling which leads to the increased permeability and solute leakage. Several postharvest techniques have been demonstrated to be effective in managing chilling injury in vegetables, including temperature preconditioning, intermittent warming, high oxygen atmosphere, heat treatment, modified atmosphere packaging, and treatment with growth regulators or natural products as well as harvesting at proper maturity. Treatment with certain naturally occurring compounds such as methyl jasmonate (MJ) or methyl salicylate (MS) increased activities of alternative oxidase (AOX) in the respiratory pathway of sweet pepper and enhanced amounts of high and low molecular weight heat shock proteins (HSP) and pathogenesis-related proteins (PRP) in tomatoes. Temperature preconditioning treatment elevated antioxidant content and antioxidant enzyme activities, and augmented s-adenosylmethionine (SAM) decarboxylase activities and increased the levels of spermidine and spermine in zucchini squash. These defense responses induced by these treatments might be part of the mechanisms that protected and prevented tissues from chilling injury. Promising techniques and new methods such as genetic engineering and transgenic skill need to be exploited for better management of chilling injury.

Comparative proteomic and metabolomic profiling of citrus fruit with enhancement of disease resistance by postharvest heat treatment

BackgroundFrom field harvest to the consumer’s table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level.ResultsMature ‘Kamei’ Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp.ConclusionsThis study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of ‘Kamei’ Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.

Drought stress tolerance of red and white clover–comparative analysis of some chaperonins and dehydrins

The ecophysiological function linked to the contribution of the legume to the nitrogen supply of the sward, as well as its high content in pastures, make red ( Trifolim pratense) and white clover ( Trifolium repens) forage plants of economic importance in temperate regions. The study aimed to characterize the expression profiles of some important stress tolerance related proteins in red clover (cv. “Start”), and two white clover cultivars – cv. “Haifa” (medium-leafed) and cv. “Debut” (small-leafed). An attempt has been made to explain the differences in red and white clover potential to sustain unfavorable environmental conditions regarding the obtained results from protein and gene profiling. Rubisco binding protein (RBP), low molecular weight heat shock proteins (HSP), calpains (ClpA and ClpP) and dehydrins (DHN) responded significantly to drought. White clover cv. “Debut” developed the lowest leaf water deficit (WD) compared to the one detected in red clover and the medium leafed white clover cultivar “Haifa”. Immunoblot and RT-PCR showed that the small leafed white clover cv. “Debut” copes with drought stress most efficiently. Detailed gene expression analysis revealed that Y 2SK type dehydrins were subjected to alternative splicing under drought and the transcripts were strongly induced in the more drought tolerant small-leafed white clover cultivar “Debut”. A homolog sequence of Y 2K dehydrin in T. pratense (GenBank ID: JF748409) and T. repens (GenBank ID: JF748410) have been amplified and an increased transcipt levels of JF748409 were detected in T. pratense drought stressed leaves. The expression pattern of SK 2 transcripts showed that this dehydrin type has been down-regulated in both white clover cultivars under water deprivation.

Identification of seed proteins associated with resistance to pre-harvested aflatoxin contamination in peanut (Arachis hypogaea L)

BackgroundPre-harvest infection of peanuts by Aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism.ResultsThe objective of this study was to identify resistance-associated proteins in response to A. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1) and a susceptible cultivar (Yueyou 7) under well-watered condition, drought stress, and A. flavus infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to A. flavus attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant cultivar challenged by A. flavus under drought stress. A significant decrease or down regulation of trypsin inhibitor caused by A. flavus in the resistant cultivar was also observed. In addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time RT-PCR analysis.ConclusionIn summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination.

Low molecular weight heat shock protein HSP27 is a prognostic indicator in rectal cancer but not colon cancer

ObjectiveThere are currently no biomarkers in routine clinical use for determining prognosis in rectal cancer. In a preliminary proteomic study, variation in the levels of heat shock protein 27 (HSP27)...

Differential accumulation of high and low molecular weight heat shock proteins in Basmati rice (Oryza sativa L.) cultivars

The accumulation of various heat shock proteins (HSPs) and their relationship with the inbuilt cold tolerance observed in Kashmir Basmati was studied. Five Basmati rice verities (Oryza sativa), Basmati-370, Basmati-Pak, Basmati-198, Basmati-385 and Kashmir Basmati were given temperature shock of 45 and 50°C. Temperature shocks were given for 16 h in incubator preheated to 45 and 50°C and 85% relative humidity. Proteins were extracted and separated on 10% acrylamide gels with 1 mm thickness and visualized for protein fractions. Accumulation of 40 kDa HSPs were observed in all the cultivars, and 20 kDa HSPs specifically in Kashmir Basmati. Small amounts of high molecular weight HSPs were observed in un-treated (control) plants of Kashmir Basmati, and it increased considerably after heat shock. The 20 kDa HSP was only expressed in heat-treated Kashmir Basmati. Differences in the expression of heat shock proteins in the tested varieties have been described in detail.

Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis.

BackgroundMicroscopic monogonont rotifers, including the euryhaline species Brachionus plicatilis, are typically found in water bodies where environmental factors restrict population growth to short periods lasting days or months. The survival of the population is ensured via the production of resting eggs that show a remarkable tolerance to unfavorable conditions and remain viable for decades. The aim of this study was to generate Expressed Sequence Tags (ESTs) for molecular characterisation of processes associated with the formation of resting eggs, their survival during dormancy and hatching.ResultsFour normalized and four subtractive libraries were constructed to provide a resource for rotifer transcriptomics associated with resting-egg formation, storage and hatching. A total of 47,926 sequences were assembled into 18,000 putative transcripts and analyzed using both Blast and GO annotation. About 28–55% (depending on the library) of the clones produced significant matches against the Swissprot and Trembl databases. Genes known to be associated with desiccation tolerance during dormancy in other organisms were identified in the EST libraries. These included genes associated with antioxidant activity, low molecular weight heat shock proteins and Late Embryonic Abundant (LEA) proteins. Real-time PCR confirmed that LEA transcripts, small heat-shock proteins and some antioxidant genes were upregulated in resting eggs, therefore suggesting that desiccation tolerance is a characteristic feature of resting eggs even though they do not necessarily fully desiccate during dormancy. The role of trehalose in resting-egg formation and survival remains unclear since there was no significant difference between resting-egg producing females and amictic females in the expression of the tps-1 gene. In view of the absence of vitellogenin transcripts, matches to lipoprotein lipase proteins suggest that, similar to the situation in dipterans, these proteins may serve as the yolk proteins in rotifers.ConclusionThe 47,926 ESTs expand significantly the current sequence resource of B. plicatilis. It describes, for the first time, genes putatively associated with resting eggs and will serve as a database for future global expression experiments, particularly for the further identification of dormancy related genes.

Microarray Analysis Reveals S-Adenosylmethionine (SAM) Synthetase Involvement in Salt Tolerance of Cyanidioschyzon merolae

We searched for candidate genes for producing salt tolerant plants from the red alga Cyanidioschyzon merolae, which lives in an extreme environment (hot springs). Arabidopsis thaliana plants die under 0.1 M salt culture, whereas the red algal cells survived under 0.3 M salt for 7 d. However, their chloroplasts changed from green to white and they soon died under 0.4 M, which is the concentration of seawater. Genes that were selectively expressed at 2 h and 24 h in 0.3 M salt concentrations were examined by microarray analysis. Under salt stress, the numbers of highly expressed genes at 2 h increased from 70 to 95 after culture for 24 h. The highly expressed genes included those encoding proteins similar to low molecular weight heat shock proteins, heat shock protein 70, and S-adenosylmethionine (SAM) synthetase. On the base of the present data and on the known metabolic functions of the proteins, we suggest that the SAM synthetase gene from C. merolae is a candidate gene for genetic engineering to produce salt tolerance plants.

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.

Effect of thermal stress on early and late passaged mouse lens epithelial cells

Cataract is an age related disease of protein aggregation. It has been suggested that aging affects the cells ability to protect protein integrity. The protein integrity, which is essential for cellular homeostasis, is maintained by a complex system of refolding or degradation of damaged proteins. The heat shock proteins (hsps) are the major contributors in the maintenance of protein integrity. The heat shock transcription factor (HSF-1) is the master regulator of all hsp synthesis in response to stress. This investigation examined the role of HSF-1 in the regulation of hsp synthesis in early and late passaged alphaTN-4 cells. Data collected in this study revealed that the nucleotide sequence of HSF-1 mRNA obtained from early and late passaged alphaTN-4 cells were identical. When early and late passaged cell were exposed to thermal stress, their hsp expression were also similar. HSP-40 expression was detected after 2 h of heat stress, whereas HSP-70 and low molecular weight heat shock protein alphabeta crystallin showed significantly increased synthesis 18 h post heat stress. The late passaged alphaTN-4 cells ability to upregulate hsps in response to heat stress could be due to its high replicative activities. The data presented here suggests a relationship between the presence of functional HSF-1 and sustained proliferative activities of the late passaged alphaTN-4 cell.

The effect of heat stress and cadmium ions on the expression of a small hsp gene in barley and maize

Small heat shock proteins in plants are produced under stress conditions and play an important role in stress tolerance. The gene Hvhsp17 isolated from barley encodes a class I, low molecular weight heat shock protein (or sHSP) which is induced in barley seedlings in response to heat stress. Previous molecular analysis of the 5′ promoter region of Hvhsp17 uncovered several cis regulatory elements upstream from the ATG of the gene, two heat shock elements (HSE1 and HSE2), and a sequence highly homologous to a metal responsive element of mammalian cells. The importance of the protective role of these elements against abiotic stresses was investigated both in barley and in maize. The expression profile of Hvhsp17 in response to various environmental conditions was analysed in these two cereals, to understand the regulation of Hvhsp17 gene expression and also in relation to conditions other than heat shock. The expression of Hvhsp17 in both barley and maize is strictly associated with heat stress, except for treatment with cadmium ions.

Direct association and translocation of PKC-alpha with calponin.

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-alpha and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-alpha, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-alpha in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.

Ubiquitin Activity Following Forebrain Ischemia/Reperfusion in the Rat

Domorakova I., E. Mechirova, J. Burda, M. Ferikova: Ubiquitin Activity Following Forebrain Ischemia/Reperfusion in the Rat. Acta Vet. Brno 2004, 73: 45-49. Cerebral ischemia/reperfusion leads to selective neuronal death in specific brain areas. The effects of forebrain ischemia produced by four-vessel occlussion were investigated in rats. Immunohistochemical localization of ubiquitin in the brain cortex and hippocampus was studied. Using antibodies against ubiquitin we have found that ubiquitin immunoreactivity (UIR) was normally present in all neurons of hippocampus and in the cerebral cortex. After 20 min of ischemia and 24 h of reperfusion the number of ubiquitin positive cells was decreased in CA2 and CA3 regions of hippocampus and ubiquitin-negative neurons were found in the most vulnerable region of CA1 subfield. Cerebral cortex showed significant changes of ubiquitin immunoreactivity in the nuclei and perikarya of pyramidal neurons. Seven days of reperfusion after 20 min of ischemia showed recovery of UIR in the hippocampal CA2, CA3 regions and in granule cells layer of the dentate gyrus. The most vulnerable CA1 pyramidal cells showed morphological changes of the cell bodies and irregularities of the cell membrane. Interestingly, number of those cells display intensive UIR in their perikarya and in the nuclei as well . Comparison of UIR in CA1 pyramidal cells after ischemia/reperfusion with those in the control sections showed increase in ubiquitin reaction. Remarkable UIR was also found in all layers in the cerebral cortex. These data suggest that recovery of ubiquitin immunoreactivity in some regions of hippocampus and cerebral cortex may be a prerequisite to neuronal survival after ischemia /reperfusion. In CA1 region the accumulation of ubiquitinated proteins in pyramidal cells was visualized without their degradation. The stress protein ubiquitin is one of the regulatory proteins playing a role in ischemic tolerance. Ischemia, hippocampus, cerebral cortex, ubiquitin, rat Specific neuronal populations in the brain are selectively vulnerable to ischemic insults. Ischemic cell death is characterized by a delay between the insult and manifestation of major cell damage. This delay varies greatly, depending on the nature of the insult and of the brain region being affected (Lipton 1999). Two different types of cell death can be discerned. One type progresses rapidly over a period of hours whereas the second type requires several days to mature (Kirino 1982). The hippocampus is a brain structure displaying both these features. Following transient periods of cerebral ischemia in the rat a rapid degeneration of the neurons in the dentate gyrus takes place. The CA1 neurons, on the other hand, show light microscopical signs of degeneration during the first days following the insult, demonstrating delayed neuronal death (Kirino 1982; Pulsinelli and Brierley 1982). Although total protein synthesis of the brain during ischemia/reperfusion is severely inhibited ( Araki et al. 1990), synthesis of specific proteins, such as heat shock proteins (HSP70) is induced. Protective role of HSP in ischemic tolerance has been reported by Kirino et al. (1991) and Liu et al. (1992). Ubiquitin, a low molecular weight heat shock protein found in all eukaryotic cells, is bound to short-lived and denatured proteins produced by various forms of injury. Ubiquitin

Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells.

Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3-like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for alpha-synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA-induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for alpha-synuclein. Time-dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by alpha-methyl-p-tyrosine (alphaMpT), or administering the antioxidant S-apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when alphaMpT-treated cells were loaded with l-DOPA, which restored intracellular dopamine levels.

An expressed sequence tag (EST) set from Citrus sinensis L. Osbeck whole seedlings and the implications of further perennial source investigations

Compared with the large-scale single pass cDNA sequencing entries from annual plants, the NCBI database has very little sequence information from perennial plant species. Although similar to annuals in many biochemical pathways, perennials are unique in the fact that they posses long generation times. Without short cycle reproduction as an escape mechanism, perennials have evolved alternative survival mechanisms to pathogen attack and environmental stresses. The study of these alternate strategies by way of functional genomics will greatly increase the understanding of the biochemical changes underlining stress responses in perennials. Herein we analyze a set of expressed sequence tags (ESTs) produced from a 180-day-old whole immature sweet orange citrus seedling cDNA library. From this library, 7680 cDNAs were single pass sequenced from the 5′ end, generating 6443 high quality ESTs. In the analysis, 2272 ESTs (35%) were found to significantly match ( E-value≤10 −10) proteins with known function in the public databases using blastx. Additionally, 1457 ESTs (23%) significantly matched proteins with unknown function and 1619 ESTs (25%) matched to proteins described as putative. The remaining 1095 ESTs (17%) failed to match with significance to any protein sequence found in the public databases. ESTs matching to the photosynthetic proteins chlorophyll A/B binding protein, plastocyanin and ribulose-1,5-bisphosphate carboxylase were abundant, 6.0% of the total. Interestingly, stress related proteins such as low molecular weight heat shock proteins, peroxidases, lipid transfer proteins and metallothionein-like proteins were also abundant, 3.6% of the total, suggesting a role for these genes in citrus seedling development .