HSPA

T he heat shock protein response appears to be triggered primarily by nonnative proteins accumulating in a stressed cell and results in increased expression of heat shock proteins (HSPs). Many heat shock proteins prevent protein aggregation and participate in refolding or elimination of misfolded proteins in their capacity as chaperones. Even though several mechanisms exist to regulate the abundance of cytosolic and nuclear chaperones, activation of heat shock transcription factor 1 (HSFl) is an essential aspect of the heat shock protein response. HSPs and co-chaperones that are assembled into multichaperone complexes regulate HSFl activity at different levels. HSP90-containing multichaperone complexes appear to be the most relevant repressors of HSFl activity. Because HSP90-containing multichaperone complexes interact not only specifically with client proteins including HSFl but also genetically with nonnative proteins, the concentration of nonnative proteins influences assembly on HSFl of HSP90-containing complexes that repress activation, and may play a role in inactivation, of the transcription factor. Proteins that are unable to achieve stable tertiary structures and remain chaperone substrates are targeted for proteasomal degradation through polyubiquitination by co-chaperone CHIP. CHIP can activate HSFl to regulate the protein quality control system that balances protection and degradation of chaperone substrates.

The low molecular weight (LMW) heat shock protein (HSP) gene hsp16.6 was identified and cloned from the unicellular cyanobacterium Synechocystis sp. PCC 6803 through comparisons of genomic sequences and conserved gene sequences of the LMW HSPs. Hsp16.6 was isolated using PCR and cloned into the pGEMT plasmid. Hsp16.6 showed a significant increase in transcription after heat shock at 42°C that indicated hsp16.6 was a heat shock gene. To determine the role that hsp16.6 plays in the heat shock response, a mutant Synechocystis cell line was generated. Cell growth and oxygen evolution rates of wild type and mutant cells were compared after heat shock. Results showed significantly decreased cell growth rates and a 40% reduction in oxygen evolution rates in mutants after heat shock treatments. These data indicate a protective role for hsp16.6 in the heat shock response.

The low molecular weight (LMW) heat shock protein (HSP) gene hsp16.6 was identified and cloned from the unicellular cyanobacterium Synechocystis sp. PCC 6803 through comparisons of genomic sequences and conserved gene sequences of the LMW HSPs. Hsp16.6 was isolated using PCR and cloned into the pGEMT plasmid. Hsp16.6 showed a significant increase in transcription after heat shock at 42°C that indicated hsp16.6 was a heat shock gene. To determine the role that hsp16.6 plays in the heat shock response, a mutant Synechocystis cell line was generated. Cell growth and oxygen evolution rates of wild type and mutant cells were compared after heat shock. Results showed significantly decreased cell growth rates and a 40% reduction in oxygen evolution rates in mutants after heat shock treatments. These data indicate a protective role for hsp16.6 in the heat shock response.

Resumo Este artigo apresenta um demonstrador de redes heterogénea desenvolvido tendo por base o projecto Europeu Moby Dick. O demonstrador suporta qualidade de serviço, mobilidade e autenticação de uma forma integrada. O artigo discute diversos aspectos da implementação do demonstrador, bem como alguns pacotes de softwaredesenvolvidos. O artigo apresenta ainda testes realizados sobre o demonstrador.

One of the most important multimedia applications is Internet protocol TV (IPTV) for next-generation networks. IPTV provides triple-play services that require high-speed access networks with the functions of multicasting and quality of service (QoS) guarantees. Among 4G mobile access networks, LTE networks are regarded as among the best solutions to meet higher bandwidth demands. In this paper, we propose a new architecture for multicasting live IPTV traffic in 4G LTE networks. The proposed mechanism involves assigning a unique logical link identifier to each IPTV channel. To manage multicasting, a prior storing server in the Base Station and in each mobile network unit, mobile phone, is constructed. In this work, we propose a partial prior storing strategy that considers the changes in the popularity of the video content segments over time and the access patterns of the users to compute the utility of the objects in the prior storage. We also propose to partition the prior storage to avoid the eviction of the popular objects (those not accessed frequently) by the unpopular ones which are accessed with higher frequency. The popularity distribution and ageing of popularity are measured from two online datasets and use the parameters in simulations. Simulation results show that our proposed architecture can improve the system performance and QoS parameters in terms of packet delay, jitter and packet loss.

Heat-resistant variants of the Chinese hamster HA-1 line have been isolated after repeated heat treatments. The heat-resistant phenotype has been stable for over 70 passages. One of the members of the 70-kDa heat shock protein family was found to be synthesized at greater levels in the heat-resistant variants under normal growth conditions. Mild heat treatment of the variant lines induced a transient thermotolerance that was accompanied by additional increase in the synthesis of the 70-kDa heat shock proteins. Cell-free translation of total cellular RNA revealed greater amounts of 70-kDa heat shock protein mRNA in both control and heated variant cells. The greater levels of 70-kDa heat shock protein synthesized in the variant cells presumably are a reflection of altered levels of its messenger mRNA. In addition, we found that translational control plays a role in the elevated expression of heat shock proteins in heat-shocked HA-1 cells and their heat-resistant variants. The association of the heat-resistant phenotype with increased levels of a 70-kDa heat shock protein suggests strongly that this gene product plays a role in protecting cells from damage inflicted by elevated temperatures.

Heat shock-related stress proteins present in all eucaryotes and procaryotes have been shown to be immune targets in a broad range of infections. We have analyzed sera from people exposed primarily to Plasmodium falciparum for specific antibodies against two heat shock-related proteins (proteins similar to the heat shock protein with a molecular weight of 75,000 [Pfhsp] and a glucose-regulated protein with a molecular weight of 72,000 [Pfgrp]). In an immunoprecipitation analysis with metabolically labeled parasites and synthetic peptides in an enzyme-linked immunosorbent assay, specific antibodies against Pfhsp and Pfgrp were detected in the sera of these individuals. Sera from people exposed to a different human malarial parasite, Plasmodium vivax, did not react with the peptides in an enzyme-linked immunosorbent assay. Southern blot analysis with DNA isolated from P. falciparum from different geographical locations showed a conservation of genes for these stress proteins; thus, th...

Genes homologous to heat shock protein 70 have been described in parasitic protozoa. It has been proposed that they may be important to the parasite as it moves from the vertebrate host at 37 degrees C to the insect. We now describe a genomic DNA clone isolated from Plasmodium falciparum that encodes a protein similar in sequence to a mammalian heat shock-related protein, the 78-kDa glucose-regulated protein of rat and hamster. The gene is expressed during the erythrocytic stage in both asexual and sexual parasites (RNA blot analysis) and a 72-kDa protein is immunoprecipitated from erythrocytic stage parasites. Importantly, the sequence of the clone is similar to the canonical sequence at the carboxyl termini of glucose-regulated proteins of mammals that determines their localization within endoplasmic reticulum. Since the parasite sequence has only three (Asp-Glu-Leu) of the four carboxyl-terminal amino acids, its location and its function within the parasite remain to be determined.

Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we sh...

Transcriptional regulation of the human hsp70 gene in response to heat shock and other forms of physiological stress occurs through the activation of heat shock transcription factor (HSF). Exposure of cells to a heat shock temperature of 42 degrees C results in transient activation of HSF; its DNA-binding activity increases rapidly, plateaus, and attenuates, during which the intracellular levels of hsp70 increase. In an effort to understand whether HSF is regulated negatively by hsp70, we have examined whether HSF associates with hsp70. We show that activated HSF associates with hsp70 and that the interaction is detected as the levels of hsp70 increase in the cell. Addition of ATP and other hydrolyzable nucleotides results in the dissociation of hsp70 from HSF while nonhydrolyzable nucleotide analogs do not disrupt the complex. We demonstrate that exogenous recombinant wild-type hsp70 can associate with activated HSF, whereas no association is observed with an amino-terminal or a ca...

Background: The AGT gene is gene responsible for regulation of protein called angiotensinogen which regulates blood pressure and balances fluids in the body. Hypertension happens due to many causes one of this is the defect in AGT gene. Hypertension usually has no symptoms. However, it is a major risk factor for heart diseases, stroke, kidney failure, and eye problems. Objectives: in this study we use software to analyze the gene using different software and represented statistically and to detect the SNPs that can cause the disease. Material and Method: In this analysis using many software tools that can analyze the nsSNPs retrieved from NCBI website. These software include SIFT, I-mutant, Polyphen-2, PHD SNP and SNP& Go, Projecthop and GeneMANIA. Results: The study showed that from 172 nsSNPs only 46 nsSNPs were deleterious while 126 were tolerated using SIFT. Two were benign, 11 were possibly damaging and 33 were probably damaging by Polyphen-2. Using Provean, 19 nsSNPs were neutral and 27 were deleterious. For PHD-SNP software 20 nsSNPs were disease related and 18 were neutral. Also SNPs were checked using SNP & Go software that showed 32 neutral nsSNPs and 14 nsSNPs were disease associated variants. Using I-Mutant software 13 nsSNPs increase the stability of the protein and 33 decrease the protein stability. Conclusions: In conclusion, extensive functional and structural analyses are carried out to predict potentially damaging and deleterious nsSNPs of AGT gene using bioinformatics and computational methods. In the study, 14 high confidence damaging nsSNPs are identified from 172 nsSNPs. Although bioinformatics tools have their limitations, the results from the present study may be convenient in future for further population based research activities and towards development of accuracy medicines.

Drosophila melanogaster tissues carrying a third chromosome with the deletion Df ( 3R ) Kar D2 make a 40,000-dalton (Dal) heat shock protein not made by wild type. The unusual polypeptide was inducible in every tissue examined. Tryptic peptide fingerprints showed it to include part of the 70,000-Dal major heat shock protein. Mapping experiments placed the mutation responsible for the 40,000-Dal protein at or close to the kar D2 deletion. One break point of the deletion is in subdivision 87A, close to or at a heat shock locus that codes for the 70,000-Dal protein. The results are consistent with the possibility that this break point is within a gene for the 70,000-Dal protein, leaving only the initial portion of its coding sequence. This would specify the direction of transcription of the mutant gene as proximal to distal on the normal chromosome. The 87A heat shock locus should contain at least two genes for the 70,000-Dal protein, because embryos homozygous for the kar D2 deletion ...

The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) is activated under different conditions including heat shock. By using transient transfection assays, we have compared the thermal activation of HIV-1 LTR to that of the promoter of the gene encoding the human stress protein hsp70 which is under the control of the heat shock transcription factor HSF. In these assays, the chloramphenicol acetyl transferase (Cat) gene was used as a reporter gene. Several parameters of the heat stress were analyzed such as the temperature, the duration of heat stress and that of the recovery period. Under every condition tested, we have found that the kinetics of activation of both promoters were very similar. In addition, both showed a similar inhibition by actinomycin D. These results were compared to those obtained with a DNA construct containing the early promoter of SV-40 virus coupled to the Cat gene. In this case, no heat-mediated accumulation of CAT protein was observed, indicating that the transcriptional activation of HIV-1 LTR by heat shock is specific. HIV-1 LTR contains two NF-tcB binding elements, involved in the activation of this promoter during oxidative stress, which are sequence related to the heat shock element HSE. However, under all the heat shock conditions tested, we have been unable to detect the binding of any protein to xB dements, suggesting that this site is not directly involved in the thermal activation of HIV-1 LTR. These results indicate that the thermal transcriptional activation of HIV-I LTR and hsp70 promoters occurs through different mechanisms that are triggered by similar heat shock conditions.

The recently described protein denoted H11, Hsp22 or HspB8 seems to participate in regulation of proliferation, apoptosis, and cardiac hypertrophy. Mutation of Hsp22 causes distal motor neuropathy. Multitude action of Hsp22 is supposed to be due to its protein kinase and/or chaperone-like activities. There are many indirect evidences indicating that Hsp22 possesses intrinsic protein kinase activity. However, low homology to protein kinases, low extent of autophosphorylation, lack of significant protein kinase activity with commonly used substrates, and lack of information on stoichiometry, kinetics, and substrate specificity make the existence of intrinsic protein kinase activity of Hsp22 questionable. It is supposed that protein kinase activity ascribed to Hsp22 is due to contaminating protein kinases. Hsp22 is highly homologous to small heat shock proteins and effectively prevents aggregation of denatured protein both in vitro and in vivo. Therefore, it is supposed that chaperone-like activity is of great importance for Hsp22 functioning.

The recently described protein denoted H11, Hsp22 or HspB8 seems to participate in regulation of proliferation, apoptosis, and cardiac hypertrophy. Mutation of Hsp22 causes distal motor neuropathy. Multitude action of Hsp22 is supposed to be due to its protein kinase and/or chaperone-like activities. There are many indirect evidences indicating that Hsp22 possesses intrinsic protein kinase activity. However, low homology to protein kinases, low extent of autophosphorylation, lack of significant protein kinase activity with commonly used substrates, and lack of information on stoichiometry, kinetics, and substrate specificity make the existence of intrinsic protein kinase activity of Hsp22 questionable. It is supposed that protein kinase activity ascribed to Hsp22 is due to contaminating protein kinases. Hsp22 is highly homologous to small heat shock proteins and effectively prevents aggregation of denatured protein both in vitro and in vivo. Therefore, it is supposed that chaperone-like activity is of great importance for Hsp22 functioning.

Untagged recombinant human small heat shock protein with apparent molecular mass 22 kDa (Hsp22) was obtained in homogeneous state. Size exclusion chromatography and chemical crosslinking with dimethylsuberimidate indicate that Hsp22 forms stable dimers. Being highly susceptible to oxidation Hsp22 forms disulfide crosslinked dimers and poorly soluble high molecular mass oligomers. According to CD spectroscopy oxidation of Hsp22 results in disturbing of both secondary and tertiary structure. Hsp22 possesses a negligibly low autophosphorylation activity and under the conditions used is unable to phosphorylate casein or histone. Hsp22 effectively prevents heat-induced aggregation of yeast alcohol dehydrogenase and bovine liver rhodanese with chaperone activity comparable to that of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20).

ABSTRACTAn open reading frame in the genomic database ofMycobacterium tuberculosisH37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned intoEscherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to t...

Nitrate reductase (NR) is the key rate-limiting enzyme of the nitrogen (N) assimilation process in plants, which has not been characterized in bread wheat under nitrogen stress, especially with respect to their homeologues. Total 9 NRs were identified and classified into 3 groups, which showed a close relationship with different wheat ancestors. The occurrence of N-responsive cis-acting regulatory elements like MYB, MYC, G-Box and GATAmotif confirmed their N-responsiveness. Expression of all the three groups of NR under N-stress revealed NR 6-1ABD group to be the most N-responsive, which was characterized further in detail. The study was carried out in two genotypes contrasting for their N-responsiveness (HD 2967: Highly responsive to applied N, and Choti Lerma: Less responsive to applied N) selected on the basis of field evaluation. Homeologous differences within a genotype were found much more than the genotypic differences of a specific homeologue coding sequence. Among the three homeologues, though NR 6-1D homeologue was found most responsive to N-stress, the contribution was maximum for this homeologue followed by NR 6-1A and least by NR 6-1B. We found that the expression of homeologues was linked to the presence of N-responsive cis-elements. All the homeologues of NR 6-1 in Choti Lerma were found less responsive to N-stress, in comparison to HD 2967, which might also be linked to N-use efficiency. Homeologous expression of NR 6-1ABD revealed negligible contribution of Bhomeologue to N-stress. Homeologous differences of NR 6-1ABD was found much more than the genotypic differences. Hence, our study on wheat NR will be helpful in manipulating the specific homeologue of the NR gene in the future.

In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [ 35 S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMPdependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, α enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.

This deliverable defines and compile the different scenarios gathered in six use cases that are relevant for the project as part of the supply chain ecosystem. For each use case, partners have defined explicit requirements and KPIs (both network and service related) that will be targeted on the development phase.

Heat shock transcription factors (HSFs) mediate the inducible transcriptional response of genes that encode heat shock proteins and molecular chaperones. In vertebrates, three related HSF genes (HSF1 to -3) and the respective gene products (HSFs) have been characterized. We report the cloning and characterization of human HSF4 (hHSF4), a novel member of the hHSF family that shares properties with other members of the HSF family yet appears to be functionally distinct. hHSF4 lacks the carboxyl-terminal hydrophobic repeat which is shared among all vertebrate HSFs and has been suggested to be involved in the negative regulation of DNA binding activity. hHSF4 is preferentially expressed in the human heart, brain, skeletal muscle, and pancreas. Transient transfection of hHSF4 in HeLa cells, which do not express hHSF4, results in a constitutively active DNA binding trimer which, unlike other members of the HSF family, lacks the properties of a transcriptional activator. Constitutive overe...

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 39-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures.

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.

An antibody highly specific for heat-shock protein (hsp)26, the unique small hsp of yeast, and mutants carrying a deletion of the HSP26 gene were used to examine the physical properties of the protein and to determine its intracellular distribution. The protein was found in complexes with a molecular mass of >500 kD. Thus, it has all of the characteristics, including sequence homology and induction patterns, of small hsps from other organisms. When log-phase cells growing in glucose were heat shocked, hsp26 concentrated in nuclei and continued to concentrate in nuclei when these cells were returned to normal temperatures for recovery. However, hsp26 did not concentrate in nuclei under a variety of other conditions. For example, in early stationary-phase cells hsp26 is induced at normal growth temperatures. This protein was generally distributed throughout the cells, even after heat shock. Similarly, in cells genetically en

The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 130 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp, morrisoni (PG14), which is also highly active against mosquito larvae.

The cells of Helicobacter pylori were suspended in the medium containing35S‐methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N‐terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.

A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 con rmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione Stransferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders.

The family of small heat shock proteins (sHsp) is composed of 10 members in mammals, four of which are found mutated in diseases associated with the accumulation of protein aggregates. Though many sHsp have demonstrated molecular chaperone activity in vitro in cell-free conditions, their activity in vivo in the normal cellular context remains unclear. In the present study, we investigated the capacity of the sHsp, HspB8/Hsp22, to prevent protein aggregation in the cells using the polyglutamine protein Htt43Q as a model. In control conditions, Htt43Q accumulated in perinuclear inclusions composed of SDS-insoluble aggregates. Co-transfected with Htt43Q, HspB8 became occasionally trapped within the inclusions; however, in most cells, HspB8 blocked inclusion formation. Biochemical analyses indicated that HspB8 inhibited the accumulation of SDS-insoluble Htt43Q as efficiently as Hsp40 which was taken as a positive control. Htt43Q then accumulated in the SDS-soluble fraction, provided that protein degradation was blocked by proteasome and autophagy inhibitors. In contrast, the other sHsp Hsp27/HspB1 and aB-crystallin/HspB5 had no effect. This suggested that HspB8 functions as a molecular chaperone, maintaining Htt43Q in a soluble state competent for rapid degradation. Analyses of Hsp27-HspB8 chimeric proteins indicated that the C-terminal domain of HspB8 contains the specific sequence necessary for chaperone activity. Missense mutations in this domain at lysine 141, which are found in human motor neuropathies, significantly reduced the chaperone activity of the protein. A decrease in the HspB8 chaperone activity may therefore contribute to the development of these diseases.

The nuclear gene for a mitochondrial ribosomal protein, termed YMR26, of Saccharomyces cerevisiae strain DC-5 was cloned by hybridization with synthetic oligonucleotide mixtures corresponding to the Nterminal amino acid sequence of this protein. The gene was found to occur in a single copy on either chromosome VII or chromosome XV. The nucleotide sequence of the cloned segment containing this gene showed the presence of an open reading frame capable of encoding a basic protein of 18.5 kDa with 158 amino acid residues. The deduced amino acid sequence showed no significant similarity to any known ribosomal proteins of prokaryotic or eukaryotic origin or to any other proteins in the NBRF protein data bank. When the gene was disrupted by insertion of a 2.9 kb restriction fragment containing LEU2, cells became PET-indicating that the gene is essential for yeast mitochondria. Northern blot analysis indicated that the size of the transcript from the YMR26 gene was approximately 530 nucleotides long. The expression level of the YMR26 gene was monitored upon catabolite repression, in strains with various mitochondrial genetic backgrounds and in strains harboring an increased dosage of the YMR26 gene. In p + cells, the transcription of the YMR26 gene was more repressed in a medium with glucose than in the presence of either galactose or nonfermentable carbon sources. However, in pO cells, its transcription appeared not to be repressed even by high concentrations of glucose. The amount of the YMR26 mRNA was increased 10-fold when cells carried the YMR26 gene on a high-copy number plasmid.

In order to understand the influence of the 20 kDa protein on the cytolytic activity of CytA protein, a set of PCR primers were designed to amplify 20 kDa protein and CytA protein genes. The genes were ligated to E. coli expression vector pUHE24 and transformed into E. coli XL1 and DH5 alpha. The clones, LZ20, LZcytA and LZ20A containing 20 kDa protein gene, cytA protein gene and 20 kDa-cytA genes were obtained respectively. The influence of the clones on the growth of E. coli cells was determined under induction of IPTG. The results showed that the growth of LZ20 cells were not affected, LZcytA cells were killed, and the growth of LZ20A cells were not affected. It was suggested that expression product of 20 kDa protein gene protected the host cells from the cytolytic effect of CytA protein. This was supported by using different host strains.

The present study examined time-dependent changes in the gene expression profile of long-term cultured human myotubes. Microarray transcriptional analysis was performed in a primary culture of differentiated myotubes from one subject over seven weeks. This analysis showed a main gradual fall in genes of the contractile apparatus, and a broad upregulation of genes involved in cell development and growth, followed by stress response and signal transduction. Glucose metabolism was also monitored, but no significant alterations in glucose uptake, oxidation or glycogen storage were observed. Mitochondrial membrane potential, or the amount of membrane lipid peroxides, remained similarly unchanged, nor was lactate dehydrogenase leakage observed. Time-dependent changes in eight genes were validated by real-time RT-PCR in primary cultured myotubes from four subjects, of similar age and isolated after equivalent replication cycles in vitro and differentiated over seven weeks. Insulinlike growth factor-binding protein 2 (IGFBP2), a modulator of the IGF signal, was upregulated. The antiapoptotic gene heat-shock 70-kd protein 2 (HSPA2) was induced, whereas the proapoptotic tumor necrosis factor receptor superfamily, member 25 (WSL-1) was suppressed. A decline in the muscle-specific gene M-cadherin and contraction genes, such as slow-twitch troponin I (TNNI1) and myosin heavy chain 2 (MYH2), myosin light chain 1 (MYL1) and myosin-binding protein H (MYBPH), which are expressed in adult fast-twitch muscle, was shown. In summary, these data demonstrate extensive downregulation of contractile genes and modulation of apoptosis-related genes, in favour of cell survival, during maintenance of cultured human myotubes.

Цель работы: разработка метода, позволяющего обнаруживать и идентифицировать пакеты сети Tor, в том числе, обфусцированных пакетов на локальной машине пользователя сети, сниффером Wireshark с использованием синтаксиса фильтров, основанных на признаках пакетов сети Tor, характерных для версии шифрования TLS v1.2 и v1.3; изучение возможности использования атаки SSL Bump (расшифровка https-трафика на виртуальном сервере при помощи самоподписываемых сертификатов x.509) для преодоления обфускации пакетов сети Tor. Метод: применялся программный анализ передаваемых сетевых пакетов, декомпозиция содержимого пакетов данных по признакам их размеров и принадлежности к протоколам шифрования, сравнительный метод в отношении различных версий протокола шифрования и ресурсов, синтез правил фильтрации на основе синтаксиса анализатора. Полученные результаты: разработан прикладной метод, позволяющий обнаруживать и идентифицировать пакеты сети Tor, в том числе, обфусцированных пакетов на локальной машине пользователя сети, сниффером Wireshark на основе синтаксиса фильтрации, опирающегося на признаки пакетов шифрования версии TLS v1.2 и v1.3; получены данные о невозможности использовать атаку SSL Bump для преодоления обфускации сети Tor.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

A novel human transcript, C9orf19, mapped to the genomic region involved in hereditary inclusion body myopathy (IBM2) at chromosome 9p12-p13, has been cloned and characterized. A single cDNA clone consisting of the full-length 1.9 kb transcript has been isolated from a human placenta cDNA library and further analyzed. Genomic characterization of the C9orf19 gene identified five exons extending over 27.2 kb of genomic DNA, located 12 kb centromeric to the tumor suppressor RECK gene. C9orf19 mRNA is expressed in a wide range of adult tissues as a single transcript, most abundantly in lung and peripheral blood leukocytes. The predicted protein contains the SCP-like extracellular protein signature classified to IPR001283, a family of evolutionary related proteins with extracellular domains, which includes the human glioma pathogenesis-related protein (GliPR), the human testis specific glycoprotein (TPX-1), and several other extracellular proteins from rodents (SCP), insects venom allergens (Ag5, Ag3), plants pathogenesis proteins (PR-1) and yeast hypothetical proteins. Homology searches with the deduced 154 amino acid protein sequence of C9orf19 revealed highly similar proteins in mouse, drosophila, nematode and yeast. Mutation analysis of C9orf19 in IBM2 patients excluded it as the disease causing gene and revealed four single nucleotide polymorphisms within and in the vicinity of the gene, which will certainly be useful tools to study its potential role in several human diseases mapped to chromosome 9p12-p13. Parallel to this study, the gene termed GNE, approximately 50 kb centromeric to C9orf19, was shown to be the disease causing gene in IBM2.

Heat shock protein A2 (HSPA2) is a testis-specific molecular chaperone of the 70 kDa heat shock protein (HSP70) family and reported to play a key role in spermatogenesis as well as in the remodelling of the sperm surface during capacitation. It is established that mice lacking HSPA2 gene are infertile and spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. However, its role in post fertilization events is not fully understood. Owing to the importance of HSPA2 in male reproduction, the present study is undertaken to reveal the association between genetic mutation and phenotypic variation in recurrent pregnancy loss (RPL) patients through an in silico prediction analysis. In this study, we used different computational tools and servers such as SIFT, PolyPhen2, PROVEAN, nsSNPAnalyzer, and SNPs & GO to analyse the functional consequences of the nsSNPs in human HSPA2 gene. The most damaging amino acid variants generated were subjected to I-Mutant 2.0 and ConSurf. Post-translational modifications such as phosphorylation mediated by these deleterious nsSNPs were analysed using NetPhos 2.0, and gene-gene interaction study was conducted using GeneMANIA. Finally, in-depth studies of the nsSNPs were studied through Project HOPE. The findings of the study revealed 18 nsSNPs to be deleterious using a combinatorial bioinformatic approach. Further functional analysis suggests that screening of nsSNP variants of HSPA2 that tend to be conserved and has potential to undergo phosphorylation at critical positions (rs764410231, rs200951589, rs756852956) may be useful for predicting outcome in altered reproductive outcome. The physicochemical alterations and its impact on the structural and functional conformity were determined by Project HOPE. Gene-gene interaction depicts its close association with antioxidant enzyme (SOD1) strongly supporting an inefficient oxidative scavenging regulatory mechanism in the spermatozoa of RPL patients as reported earlier. The present study has thus identified high-risk deleterious nsSNPs of HSPA2 gene and would be beneficial in the diagnosis and prognosis of the paternal effects in RPL patients.

Heat shock protein A2 (HSPA2) is a testis-specific molecular chaperone of the 70 kDa heat shock protein (HSP70) family and reported to play a key role in spermatogenesis as well as in the remodelling of the sperm surface during capacitation. It is established that mice lacking HSPA2 gene are infertile and spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. However, its role in post fertilization events is not fully understood. Owing to the importance of HSPA2 in male reproduction, the present study is undertaken to reveal the association between genetic mutation and phenotypic variation in recurrent pregnancy loss (RPL) patients through an in silico prediction analysis. In this study, we used different computational tools and servers such as SIFT, PolyPhen2, PROVEAN, nsSNPAnalyzer, and SNPs & GO to analyse the functional consequences of the nsSNPs in human HSPA2 gene. The most damaging amino acid variants generated were subjected to I-Mutant 2.0 and ConSurf. Post-translational modifications such as phosphorylation mediated by these deleterious nsSNPs were analysed using NetPhos 2.0, and gene-gene interaction study was conducted using GeneMANIA. Finally, in-depth studies of the nsSNPs were studied through Project HOPE. The findings of the study revealed 18 nsSNPs to be deleterious using a combinatorial bioinformatic approach. Further functional analysis suggests that screening of nsSNP variants of HSPA2 that tend to be conserved and has potential to undergo phosphorylation at critical positions (rs764410231, rs200951589, rs756852956) may be useful for predicting outcome in altered reproductive outcome. The physicochemical alterations and its impact on the structural and functional conformity were determined by Project HOPE. Gene-gene interaction depicts its close association with antioxidant enzyme (SOD1) strongly supporting an inefficient oxidative scavenging regulatory mechanism in the spermatozoa of RPL patients as reported earlier. The present study has thus identified high-risk deleterious nsSNPs of HSPA2 gene and would be beneficial in the diagnosis and prognosis of the paternal effects in RPL patients.

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the en...

We have previously shown that the human 70-kilodalton heat shock protein gene (hsp7O) is induced by the adenovirus ElA gene product and during the S-G2 phase of the cell cycle. In this study, we investigated the effect of ElA on the expression of other human hsp genes. A gene encoding one form of the hsp89 protein (hsp89a) was activated during an adenovirus infection with kinetics similar to those of activation of hsp7O. The induction required a functional ElA gene. However, the hsp89 transcript was not cell cycle regulated. Genes encoding another form of hsp89 and the hsp27 protein were not induced by ElA or during the cell cycle. Further examination of hsp7O expression revealed a greater complexity than previously seen. Si nuclease

It has recently been suggested that perhaps as many as 20% of all mitochondrial proteins are regulated through lysine acetylation while Sirt3 has been implicated as an important mitochondrial protein deacetylase. It is therefore of crucial importance that the mitochondrial localization of potential protein deacetylases is unambiguously established. While mouse Sirt3 was recently shown to be mitochondrial, human Sirt3 (HsSirt3) was reported to be both nuclear and mitochondrial and to relocate from the nucleus to the mitochondrion upon cellular stress. We show here using various HsSirt3 expression constructs and a combination of immunofluorescence and careful subcellular fractionation that in contrast to the earlier report human Sirt3 is exclusively mitochondrial. We discuss possible experimental explanations for these discrepancies. In addition, we suggest on the basis of the analysis of public genome databases that the fulllength mouse Sirt3 protein is a 37 kDa mitochondrial precursor protein contrary to the previously suggested 29 kDa protein.

After cells have been exposed to a nonlethal heat shock, they develop an enhanced resistance to subsequent prolonged heat shock. This process, termed thermotolerance, correlates with the expression of a group of proteins called the heat shock proteins. When cells are exposed to heat, protein synthesis is rapidly turned off and takes 5-6 hr to recover. In thermotolerant cells, protein synthesis is not blocked by heat. The heat shock proteins are thought to be responsible for the development of therrnotolerance and the protection of the protein synthesis machinery from heat inactivation. To test the hypothesis that the heat shock proteins are involved in the heat shock response, we used two inhibitors to block their transcription and expression during heating and then monitored the effect on the development of thermotolerance and on protein synthesis. Carnptothecin inhibits

Heat shock leads to coordinate increases in transcription of a family of heat shock genes, including the mouse hsp70.1 and B2 genes. Activation of the heat shock transcription factor (HSF) by heat shock stimulates transcription of the murine hsp70.1 gene (by RNA polymerase 11). B2 genes are short, repetitive sequences whose transcription (by RNA polymerase Ill) are also increased after heat shock. We have studied whether heat-induced transcription is auto-regulated by the products of the heat shock genes. The results indicate: (1) after an initial heat shock, transcription of the heat shock genes by RNA polymerases II and 111 becomes desensitized to further heat shock, and the heat-induced DNA binding activity of the HSF is lost, (2) if accumulation of heat shock gene products is inhibited, the desensitizing effect of a prior heat shock is removed, and (3) transcription of the hsp70.1 and B2 genes apparently involves different mechanisms, with hsp70.1 employing the HSF and the 82 gene using a separate, heat-activated transcriptional mechanism. However, the level of transcription from the hsp70.1 and B2 genes and the stability of their respective KNAs are co-ordinateiy regulated by the level of heat shock protein in the cell. The data indicate that auto-regulation of the level of mouse heat shock gene products is mediated by RNA polymerase II transcripts but that the regulatory mechanism can control transcription from RNA polymerase 111 genes as well.

After cells have been exposed to a nonlethal heat shock, they develop an enhanced resistance to subsequent prolonged heat shock. This process, termed thermotolerance, correlates with the expression of a group of proteins called the heat shock proteins. When cells are exposed to heat, protein synthesis is rapidly turned off and takes 5-6 hr to recover. In thermotolerant cells, protein synthesis is not blocked by heat. The heat shock proteins are thought to be responsible for the development of therrnotolerance and the protection of the protein synthesis machinery from heat inactivation. To test the hypothesis that the heat shock proteins are involved in the heat shock response, we used two inhibitors to block their transcription and expression during heating and then monitored the effect on the development of thermotolerance and on protein synthesis. Carnptothecin inhibits

Heat shock leads to coordinate increases in transcription of a family of heat shock genes, including the mouse hsp70.1 and B2 genes. Activation of the heat shock transcription factor (HSF) by heat shock stimulates transcription of the murine hsp70.1 gene (by RNA polymerase 11). B2 genes are short, repetitive sequences whose transcription (by RNA polymerase Ill) are also increased after heat shock. We have studied whether heat-induced transcription is auto-regulated by the products of the heat shock genes. The results indicate: (1) after an initial heat shock, transcription of the heat shock genes by RNA polymerases II and 111 becomes desensitized to further heat shock, and the heat-induced DNA binding activity of the HSF is lost, (2) if accumulation of heat shock gene products is inhibited, the desensitizing effect of a prior heat shock is removed, and (3) transcription of the hsp70.1 and B2 genes apparently involves different mechanisms, with hsp70.1 employing the HSF and the 82 gene using a separate, heat-activated transcriptional mechanism. However, the level of transcription from the hsp70.1 and B2 genes and the stability of their respective KNAs are co-ordinateiy regulated by the level of heat shock protein in the cell. The data indicate that auto-regulation of the level of mouse heat shock gene products is mediated by RNA polymerase II transcripts but that the regulatory mechanism can control transcription from RNA polymerase 111 genes as well.