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Different types of stress including heat shock may induce genomic instability, due to the derepression and amplification of mobile elements (MEs). It remains unclear, however, whether piRNA-machinery regulating ME expression functions normally under stressful conditions. The aim of this study was to explore the features of piRNA expression after heat shock (HS) exposure inDrosophila melanogaster.We also evaluated functioning of piRNA-machinery in the absence of major stress proteinHsp70in this species. We analyzed the deep sequence data of piRNA expression after HS treatment and demonstrated that it modulates the expression of certain double-stranded germinal piRNA-clusters. Notable, we demonstrated significant changes in piRNA levels targeting a group of MEs after HS only in the strain containing normal set ofhsp70genes. Surprisingly, we failed to detect any correlation between the levels of piRNAs and the transcription of complementary MEs in the studied strains. We propose that modulation of certain piRNA-clusters expression upon HS exposure inD. melanogasteroccurs due to HS-induced altering of chromatin state at certain chromosome regions. [ABSTRACT FROM AUTHOR]

In the present era of global warming and dramatically increased environmental pollution posing a threat to animal life, the understanding and manipulation of organisms' resources of stress tolerance is apparently a question of survival. Heat stress and other forms of stressful factors induce a highly organized response of organisms at the cellular level where heat shock proteins (Hsps) and in particular Hsp70 family of chaperones are among the major players in the protection from the environmental challenge. The present review article summarizes the peculiarities of the Hsp70 family of proteins protective functions being a result of many millions of years of adaptive evolution. It discusses the molecular structure and specific details of hsp70 gene regulation in various organisms, living in diverse climatic zones, with a special emphasis on the protective role of Hsp70 in adverse conditions of the environment. The review discusses the molecular mechanisms underlying Hsp70-specific properties that emerged in the course of adaptation to harsh environmental conditions. This review also includes the data on the anti-inflammatory role of Hsp70 and the involvement of endogenous and recombinant Hsp70 (recHsp70) in proteostatic machinery in various pathologies including neurodegenerative ones such as Alzheimer's and Parkinson's diseases in rodent model organisms and humans in vivo and in vitro . Specifically, the role of Hsp70 as an indicator of disease type and severity and the use of recHsp70 in several pathologies are discussed. The review discusses different roles exhibited by Hsp70 in various diseases including the dual and sometimes antagonistic role of this chaperone in various forms of cancer and viral infection including the SARS-Cov-2 case. Since Hsp70 apparently plays an important role in many diseases and pathologies and has significant therapeutic potential there is a dire need to develop cheap recombinant Hsp70 production and further investigate the interaction of externally supplied and endogenous Hsp70 in chaperonotherapy., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

The enzymes involved in the transsulfuration pathway and hydrogen sulfide production-cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - play an important cytoprotective role in the functioning of the organism. Using CRISPER/Cas9 technology, we obtained Drosophila strains with deleted cbs, cse, and mst genes as well as with double deletion of cbs and cse genes. We analyzed the effect of these mutations on the pattern of protein synthesis in the salivary glands of third instar larvae and in the ovaries of mature flies. In the salivary glands of strains with cbs and cse deletions, a decrease was found in the accumulation of the FBP2 storage protein containing 20% methionine amino acid residues. In the ovaries, changes were detected in the level of expression and isofocusing points of proteins involved in cell protection against oxidative stress, hypoxia, and protein degradation. It was shown that in the strains with deletions of transsulfuration enzymes the proteins have a similar degree of oxidation to that of the control strain. A decrease in the total number of proteasomes and their activity was found in the strains with deletions of the cbs and cse genes.

The gasotransmitter hydrogen sulfide (H 2 S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H 2 S production and metabolism. Here, we investigated the role of H 2 S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

The gasotransmitter hydrogen sulfide (H 2 S) is an important biological mediator, playing an essential role in many physiological and pathological processes. It is produced by transsulfuration - an evolutionarily highly conserved pathway for the metabolism of sulfur-containing amino acids methionine and cysteine. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in cysteine metabolism and H 2 S production. Here we investigated the fitness components (longevity, stress resistance, viability of preimaginal stages, and reproductive function parameters) in D. melanogaster lines containing deletions of the CBS and CSE genes. Surprisingly, in most tests, CSE deletion improved, and CBS worsened the fitness. Lines with deletion of both CBS and CSE demonstrated better stress resistance and longevity than lines with single CBS deletion. At the same time, deletion of both CBS and CSE genes causes more serious disturbances of reproductive function parameters than single CBS deletion. Thus, a complex interaction of H 2 S-producing pathways and cellular stress response in determining the lifespan and fitness components of the whole organism was revealed., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Prognosis of neuropsychiatric disorders in progeny requires consideration of individual (1) parent-of-origin effects (POEs) relying on (2) the nerve cell nuclear 3D chromatin architecture and (3) impact of parent-specific miRNAs. Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting, or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMPdependent, the second relies on actin remodeling by small GTPase Rac1 - LIMK1 (LIM-kinase 1). Simple experimental model systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies. Recently, we have developed a Drosophila model for Williams-Beuren Syndrome (WBS): a mutant agn ts3 of the agnostic locus (X:11AB) harboring the dlimk1 gene. The agn ts3 mutation drastically increases the frequency of ectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affects locomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between agn ts3 and the wild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal gene origin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with the occurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat. Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-term memory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Several parameters of locomotion demonstrate heterosis. Our data indicate that the agn ts3 locus is capable of trans-regulating gene activity via POEs on the chromatin nuclear organization, thereby affecting behavior., (Copyright © AUTHORS.)

Alzheimer's disease (AD) is a neurodegenerative disease that inevitably results in dementia and death. Currently, there are no pathogenetically grounded methods for the prevention and treatment of AD, and all current treatment regimens are symptomatic and unable to significantly delay the development of dementia. The accumulation of β-amyloid peptide (Aβ), which is a spontaneous, aggregation-prone, and neurotoxic product of the processing of signaling protein APP (Amyloid Precursor Protein), in brain tissues, primarily in the hippocampus and the frontal cortex, was for a long time considered the main cause of neurodegenerative changes in AD. However, attempts to treat AD based on decreasing Aβ production and aggregation did not bring significant clinical results. More and more arguments are arising in favor of the fact that the overproduction of Aβ in most cases of AD is not the initial cause, but a concomitant event of pathological processes in the course of the development of sporadic AD. The concept of neuroinflammation has come to the fore, suggesting that inflammatory responses play the leading role in the initiation and development of AD, both in brain tissue and in the periphery. The hypothesis about the key role of neuroinflammation in the pathogenesis of AD opens up new opportunities in the search for ways to treat and prevent this socially significant disease.

Heat shock proteins (Hsps) represent the most evolutionarily ancient, conserved, and universal system for protecting cells and the whole body from various types of stress. Among Hsps, the group of proteins with a molecular weight of 70 kDa (Hsp70) plays a particularly important role. These proteins are molecular chaperones that restore the native conformation of partially denatured proteins after exposure to proteotoxic forms of stress and are critical for the folding and intracellular trafficking of de novo synthesized proteins under normal conditions. Hsp70s are expressed at high levels in the central nervous system (CNS) of various animals and protect neurons from various types of stress, including heat shock, hypoxia, and toxins. Numerous molecular and behavioral studies have indicated that Hsp70s expressed in the CNS are important for memory formation. These proteins contribute to the folding and transport of synaptic proteins, modulate signaling cascades associated with synaptic activation, and participate in mechanisms of neurotransmitter release. In addition, HSF1, a transcription factor that is activated under stress conditions and mediates Hsps transcription, is also involved in the transcription of genes encoding many synaptic proteins, whose levels are increased in neurons under stress and during memory formation. Thus, stress activates the molecular mechanisms of memory formation, thereby allowing animals to better remember and later avoid potentially dangerous stimuli. Finally, Hsp70 has significant protective potential in neurodegenerative diseases. Increasing the level of endogenous Hsp70 synthesis or injecting exogenous Hsp70 reduces neurodegeneration, stimulates neurogenesis, and restores memory in animal models of ischemia and Alzheimer's disease. These findings allow us to consider recombinant Hsp70 and/or Hsp70 pharmacological inducers as potential drugs for use in the treatment of ischemic injury and neurodegenerative disorders.

Heat shock proteins, in particular Hsp70, play a central role in proteostasis in eukaryotic cells. Due to its chaperone properties, Hsp70 is involved in various processes after stress and under normal physiological conditions. In contrast to mammals and many Diptera species, inducible members of the Hsp70 family in Drosophila are constitutively synthesized at a low level and undergo dramatic induction after temperature elevation or other forms of stress. In the courtship suppression paradigm used in this study, Drosophila males that have been repeatedly rejected by mated females during courtship are less likely than naive males to court other females. Although numerous genes with known function were identified to play important roles in long-term memory, there is, to the best of our knowledge, no direct evidence implicating Hsp70 in this process. To elucidate a possible role of Hsp70 in memory formation, we used D. melanogaster strains containing different hsp70 copy numbers, including strains carrying a deletion of all six hsp70 genes. Our investigations exploring the memory of courtship rejection paradigm demonstrated that a low constitutive level of Hsp70 is apparently required for learning and the formation of short and long-term memories in males. The performed transcriptomic studies demonstrate that males with different hsp70 copy numbers differ significantly in the expression of a few definite groups of genes involved in mating, reproduction, and immunity in response to rejection. Specifically, our analysis reveals several major pathways that depend on the presence of hsp70 in the genome and participate in memory formation and consolidation, including the cAMP signaling cascade.

Background: Hydrogen sulfide donors reduce inflammatory signaling in vitro and in vivo. The biological effect mediated by H 2 S donors depends on the kinetics of the gas release from the donor molecule. However, the molecular mechanisms of H 2 S-induced immunomodulation were poorly addressed. Here, we studied the effect of two different hydrogen sulfide (H 2 S)-producing agents on the generation of the LPS-induced inflammatory mediators. Importantly, we investigated the transcriptomic changes that take place in human cells after the LPS challenge, combined with the pretreatment with a slow-releasing H 2 S donor-GYY4137., Methods: We investigated the effects of GYY4137 and sodium hydrosulfide on the release of proinflammatory molecules such as ROS, NO and TNF-α from LPS-treated human SH-SY5Y neuroblastoma and the THP-1 promonocytic cell lines. Transcriptomic and RT-qPCR studies using THP-1 cells were performed to monitor the effects of the GYY4137 on multiple signaling pathways, including various immune-related and proinflammatory genes after combined action of LPS and GYY4137., Results: The GYY4137 and sodium hydrosulfide differed in the ability to reduce the production of the LPS-evoked proinflammatory mediators. The pre-treatment with GYY4137 resulted in a drastic down-regulation of many TNF-α effectors that are induced by LPS treatment in THP-1 cells. Furthermore, GYY4137 pretreatment of LPS-exposed cells ameliorates the LPS-mediated induction of multiple pro-inflammatory genes and decreases expression of immunoproteasome genes. Besides, in these experiments we detected the up-regulation of several important pathways that are inhibited by LPS., Conclusion: Based on the obtained results we believe that our transcriptomic analysis significantly contributes to the understanding of the molecular mechanisms of anti-inflammatory and cytoprotective activity of hydrogen sulfide donors, and highlights their potential against LPS challenges and other forms of inflammation.

Heat shock proteins (HSPs) are important factors of protein homeostasis and possess chaperone properties, providing for a folding and intracellular transport of proteins and facilitating the recovery or utilization of proteins partly denatured on exposure to various stress factors. Proteins of the Hsp70 family are the most universal molecular chaperones and interact with the greatest number of protein substrates. Several proteins of the Hsp70 family are released into the extracellular space, where they play an important role in intercellular communications and act as alarmins, or "danger signals," to modulate the immune response. The secreted Hsp70 can additionally act as an effective neuroprotector, increasing the survival of neurons in various proteinopathies, as has been demonstrated in Alzheimer's and Parkinson's disease models. In this regard, recombinant Hsp70 and inducers of endogenous Hsp70 synthesis may be considered as candidate therapeutics with immune-modulating and neuroprotective properties.Heat shock proteins (HSPs) are important factors of protein homeostasis and possess chaperone properties, providing for a folding and intracellular transport of proteins and facilitating the recovery or utilization of proteins partly denatured on exposure to various stress factors. Proteins of the Hsp70 family are the most universal molecular chaperones and interact with the greatest number of protein substrates. Several proteins of the Hsp70 family are released into the extracellular space, where they play an important role in intercellular communications and act as alarmins, or "danger signals," to modulate the immune response. The secreted Hsp70 can additionally act as an effective neuroprotector, increasing the survival of neurons in various proteinopathies, as has been demonstrated in Alzheimer's and Parkinson's disease models. In this regard, recombinant Hsp70 and inducers of endogenous Hsp70 synthesis may be considered as candidate therapeutics with immune-modulating and neuroprotective properties.

The accumulation and aggregation of β-amyloids are major molecular events underlying the progression of Alzheimer's disease. In neural cells, recombinant HSP70 reduces the toxic effect of Aβ and its isomeric forms. Here we describe the proteome of the neuroblastoma cell line after incubation with amyloid peptides Aβ42 and isomerized Asp7 (isoAβ42) without and with human recombinant heat shock protein 70 (HSP70). Incubation of SH-SY5Y cell culture with the synthetic Aβ-peptides leads to a decrease in the levels of several cytoskeleton proteins (e.g., ACTN1, VIME, TPM3) and several chaperonines involved in the folding of actin and tubulin (TCPQ, TCPG, TCPE, TCPB). These changes are accompanied by an increase in the expression of calmodulin and the proteins involved in folding in the endoplasmic reticulum and endoplasmic cell stress response. The presence of exogenous HSP70 has led to an increase in expression of several chaperones and a few other proteins including endogenous HSP70. A combined effect of recombinant HSP70 with Aβ peptides reduced cell apoptosis and significantly decreased the level of tubulin phosphorylation caused by the addition of Aβ peptides.

Neuronal dysfunction and loss associated with the accumulation of amyloid-β (Aβ) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer's disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aβ42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aβ42 with isomerised Asp7 (isoAβ42) which is one of the most abundant Aβ isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAβ42 rather than intact Aβ42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAβ42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAβ42, being to date the only known synthetic Aβ species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAβ42 in AD progression and provide possible neurotoxicity paths for this particular isoform.

Heat shock proteins (Hsp70s) from two Diptera species that drastically differ in their heat shock response and longevity were investigated. Drosophila melanogaster is characterized by the absence of Hsp70 and other hsps under normal conditions and the dramatic induction of hsp synthesis after temperature elevation. The other Diptera species examined belongs to the Stratiomyidae family (Stratiomys singularior) and exhibits high levels of inducible Hsp70 under normal conditions coupled with a thermotolerant phenotype and much longer lifespan. To evaluate the impact of hsp70 genes on thermotolerance and longevity, we made use of a D. melanogaster strain that lacks all hsp70 genes. We introduced single copies of either S. singularior or D. melanogaster hsp70 into this strain and monitored the obtained transgenic flies in terms of thermotolerance and longevity. We developed transgenic strains containing the S. singularior hsp70 gene under control of a D. melanogaster hsp70 promoter. Although these adult flies did synthesize the corresponding mRNA after heat shock, they were not superior to the flies containing a single copy of D. melanogaster hsp70 in thermotolerance and longevity. By contrast, Stratiomyidae Hsp70 provided significantly higher thermotolerance at the larval stage in comparison with endogenous Hsp70., (© 2017 The Royal Entomological Society.)

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR's target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Genomic disorders, the syndromes with multiple manifestations, may occur sporadically due to unequal recombination in chromosomal regions with specific architecture. Therefore, each patient may carry an individual structural variant of DNA sequence (SV) with small insertions and deletions (INDELs) sometimes less than 10 bp. The transposable elements of the Tc1/ mariner superfamily are often associated with hotspots for homologous recombination involved in human genetic disorders, such as Williams Beuren Syndromes (WBS) with LIM-kinase 1-dependent cognitive defects. The Drosophila melanogaster mutant agn ts 3 has unusual architecture of the agnostic locus harboring LIMK1 : it is a hotspot of chromosome breaks, ectopic contacts, underreplication, and recombination. Here, we present the analysis of LIMK1 -containing locus sequencing data in agn ts 3 and three D. melanogaster wild-type strains- Canton-S, Berlin , and Oregon-R . We found multiple strain-specific SVs, namely, single base changes and small INDEls. The specific feature of agn ts 3 is 28 bp A/T-rich insertion in intron 1 of LIMK1 and the insertion of mobile S-element from Tc1/ mariner superfamily residing ~460 bp downstream LIMK1 3'UTR. Neither of SVs leads to amino acid substitutions in agn ts 3 LIMK1. However, they apparently affect the nucleosome distribution, non-canonical DNA structure formation and transcriptional factors binding. Interestingly, the overall expression of miRNAs including the biomarkers for human neurological diseases, is drastically reduced in agn ts 3 relative to the wild-type strains. Thus, LIMK1 DNA structure per se , as well as the pronounced changes in total miRNAs profile, probably lead to LIMK1 dysregulation and complex behavioral dysfunctions observed in agn ts 3 making this mutant a simple plausible Drosophila model for WBS.

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer., Competing Interests: The authors declare they have no conflict of interest.

The genome expression pattern is strongly modified during the heat shock response (HSR) to form an adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. Here, we investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in Drosophila melanogaster We demonstrated that pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. It was also shown that deletion of all hsp70 genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR., (© 2016 The Authors.)

Heat shock protein 70 (Hsp70) is the major player that underlies adaptive response to hyperthermia in all organisms studied to date. We investigated patterns of Hsp70 expression in larvae of dipteran species collected from natural populations of species belonging to four families from different evolutionary lineages of the order Diptera: Stratiomyidae, Tabanidae, Chironomidae and Ceratopogonidae. All investigated species showed a Hsp70 expression pattern that was different from the pattern in Drosophila. In contrast to Drosophila, all of the species in the families studied were characterized by high constitutive levels of Hsp70, which was more stable than that in Drosophila. When Stratiomyidae Hsp70 proteins were expressed in Drosophila cells, they became as short-lived as the endogenous Hsp70. Interestingly, three species of Ceratopogonidae and a cold-water species of Chironomidae exhibited high constitutive levels of Hsp70 mRNA and high basal levels of Hsp70. Furthermore, two species of Tabanidae were characterized by significant constitutive levels of Hsp70 and highly stable Hsp70 mRNA. In most cases, heat-resistant species were characterized by a higher basal level of Hsp70 than more thermosensitive species. These data suggest that different trends were realized during the evolution of the molecular mechanisms underlying the regulation of the responses of Hsp70 genes to temperature fluctuations in the studied families., (© 2016 The Royal Entomological Society.)

Heat shock proteins including the major stress protein HSP70 support intracellular homeostasis and prevent protein damage after a temperature increase and other stressful environmental stimuli, as well as during aging. We have shown earlier that prolonged administration of recombinant human HSP70 to mice exhibiting Alzheimer's-like neurodegeneration as well as during sepsis reduces the clinical manifestations of these pathologies. Herein, we studied the action of recombinant human HSP70 on young and aged mouse mesenchymal stem cells (MSCs) in culture. The results obtained indicate that HSP70 at concentrations of 2 μg/ml and higher significantly stimulates growth of aged but not young MSCs. A similar effect is produced by application of a mild heat shock (42 °C 5 min) to the cells. Importantly, responses of young and aged MSCs to heat shock treatment of various durations differed drastically, and aged MSCs were significantly more sensitive to higher heat stress exposures than the young cells. Western blotting and protein labeling experiments demonstrated that neither mild heat shock nor exogenous HSP70 administration resulted in significant endogenous HSP70 induction in young and aged MSCs, whereas mild heat shock increased HSC70 levels in aged MSCs. The results of this study suggest that the administration of exogenous HSP70 and the application of mild heat stress may produce a certain "rejuvenating" effect on MSCs and possibly other cell types in vivo, and these interventions may potentially be used for life extension by delaying various manifestations of aging at the molecular and cellular level.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Microencapsulated heat shock proteins HSP 70 were studied in terms of their effects on neutrophil apoptosis, production of reactive oxygen species, and secretion of TNF-α by human neurtrophils and monocytes. Encapsulated HSP70 inhibited neutrophil apoptosis by 65% as compared to the effect of nonencapsulated HSP70; TNF-α production by the promonocytic THP-1 cells was similarly inhibited by the non-encapsulated and encapsulated HSP70. Thus, the polyelectrolyte micromolecules can be used as containers for effective delivery of HSP70 up to neutrophils and monocytes to correct the innate immunity functions.

Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.

Over the last decade, it has become evident that in mammals, including humans, heat shock protein 70 (HSP70), apart from its intracellular localization, is found in extracellular space, where it may execute various protective functions. Furthermore, the upregulation of HSP70 family members can be beneficial in the prevention and treatment of various human neurodegenerative diseases and cancer. Here, we demonstrate that recombinant human HSP70 after intranasal administration can penetrate various brain regions of mice in its native form and subsequently undergo rapid degradation. It was also shown that labeled HSP70 added to culture medium of different human and mouse cell lines enters the cells with strikingly different kinetics, which positively correlates with the basic levels of membrane bound Toll-like receptors (TLR) that are characteristic of these cell lines. HSP70 administration does not significantly modulate the level of TLR expression at the protein or RNA level. The degradation of the introduced recombinant HSP70 after entering the cells is likely proteasome-dependent and varies significantly depending on the cells type and origin. These results should be considered when developing HSP70-based therapies.

Heat shock proteins (Hsp) provide cellular and whole body adaptation of animals to various adverse environmental conditions. Hsp70 is apparently the major player underlying biological adaptation in all organisms studied so far. In all animals the regulatory regions of studied heat shock genes include several conservative promoter elements HSEs (heat shock elements) that are necessary for binding of heat shock transcription factor (HSF). The promoter regions of hsp70 genes are extremely conserved and, hence, it was generally accepted that they are universal and can operate in species belonging to different phyla. In the present work we performed the comparative analysis which revealed characteristic differences in the hsp 70 promoters of two Diptera species: Drosophila melanogaster and highly thermotolerance soldier fly Stratiomys singularior. We measured promoters activity in D. melanogaster cell culture exploring in vitro luciferase reporter assay. The analysis demonstrated significantly higher strength ofD. melanogaster promoters in spite of the fact that comparable numbers of HSEs are present in both species. These drastic differences in the promoter strength are probably due to absence of GAF-binding sites, which are necessary for efficient functioning of D. melanogaster hsp70 promoters. In contrast, comparison of hsp83 promoters isolated from these two species does not show significant differences. Our results demonstrate existence of different evolutionary trends in the regulation of the hsp70 expression even within the same order (Diptera).

Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans.

The heat shock transcription factor (HSF) is a universal activator of hsp gene expression in eukaryotes. A temperature sensitive Drosophila melanogaster strain (hsf4) with a mutation in the hsfgene was originally described as a strain lacking the transcription of hsp genes in response to heat shock. Our results demonstrated that physiological function of HSF4 is not fully abrogated after heat exposure and is able to recover even after severe heat stress, causing the induction of hsp gene expression. We have studied the kinetics of accumulation and degradation of hsp gene products at transcriptional and translational levels and shown that induction of hsp genes, particularly hsp68, in mutant strain is weaker than that in the wild type. Thus, despite the fact that the HSF4 causes a delayed ac- tivation of hsp, response to heat shock in hsf4 strain remains defective.

Background: Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats., Results: Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed., Conclusion: Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.

The heat shock proteins belonging to the Hsp90 family (Hsp83 in Diptera) play a crucial role in the protection of cells due to their chaperoning functions. We sequenced hsp90 genes from three species of the family Stratiomyidae (Diptera) living in thermally different habitats and characterized by extraordinarily high thermotolerance. The sequence variation and structure of the hsp90 family genes were compared with previously described features of hsp70 copies isolated from the same species. Two functional hsp83 genes were found in the species studied, that are arranged in tandem orientation at least in one of them. This organization was not previously described. Stratiomyidae hsp83 genes share a high level of identity with hsp83 of Drosophila, and the deduced protein possesses five conserved amino acid sequence motifs characteristic of the Hsp90 family as well as the C-terminus MEEVD sequence characteristic of the cytosolic isoform. A comparison of the hsp83 promoters of two Stratiomyidae species from thermally contrasting habitats demonstrated that while both species contain canonical heat shock elements in the same position, only one of the species contains functional GAF-binding elements. Our data indicate that in the same species, hsp83 family genes show a higher evolution rate than the hsp70 family., (© 2013 Royal Entomological Society.)

We studied various aspects of heat-shock response with special emphasis on the expression of heat-shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5-fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats., (© 2012 Blackwell Publishing Ltd.)

Although small RNAs efficiently control transposition activity of most transposons in the host genome, such an immune system is not always applicable against a new transposon's invasions. Here, we explored a possibility to introduce potentially mobile copy of the Penelope retroelement previously implicated in hybrid dysgenesis syndrome in Drosophila virilis into the genomes of two distant Drosophila species. The consequences of such introduction were monitored at different phases after experimental colonization as well as in D. virilis species, which is apparently in the process of ongoing Penelope invasion. We investigated the expression of Penelope and biogenesis of Penelope-derived small RNAs in D. virilis and D. melanogaster strains originally lacking active copies of this element after experimental Penelope invasion. These strains were transformed by constructs containing intact Penelope copies. We show that immediately after transformation, which imitates the first stage of retroelement invasion, Penelope undergoes transposition predominantly in somatic tissues, and may produce siRNAs that are apparently unable to completely silence its activity. However, at the later stages of colonization Penelope copies may jump into one of the piRNA-clusters, which results in production of homologous piRNAs that are maternally deposited and can silence euchromatic transcriptionally active copies of Penelope in trans and, hence, prevent further amplification of the invader in the host genome. Intact Penelope copies and different classes of Penelope-derived small RNAs were found in most geographical strains of D. virilis collected throughout the world. Importantly, all strains of this species containing full-length Penelope tested do not produce gonadal sterility in dysgenic crosses and, hence, exhibit neutral cytotype. To understand whether RNA interference mechanism able to target Penelope operates in related species of the virilis group, we correlated the presence of full-length and potentially active Penelope with the occurrence of piRNAs homologous to this transposable element in the ovaries of species comprising the group. It was demonstrated that Penelope-derived piRNAs are present in all virilis group species containing full-length but transcriptionally silent copies of this element that probably represent the remnants of its previous invasions taking place in the course of the virilis species divergent evolution.

Hypomorphic mutations have been investigated of the genes spineless (ss), Distal-less (Dll), leg-arista-wing complex/TBP-related factor 2(lawc/Trf2), CG5017, and hsp 70 in order to explore the effects of their expression level on the formation of distal structures of antenna and legs of Drosophila melanogaster. We demonstrated the effect of the CG5017, hsp 70, Dll, and lawc gene transcription level on phenotypic manifestation of the ss gene mutation and the involvement of these genes into morphogenesis regulation of Drosophila melanogaster limbs. The total decrease in the level of the CG5017, hsp 70, Dll, and laws gene expression level was shown to promote a loss of the segmented pattern of distal structures of legs and antennae and a reversion of Drosophila limb morphogenesis to the evolutionarily earlier progenitors of insects. A hypothesis is proposed considering limb morphogenesis of insects as an evolutionary ancient process of formation of amorphous-crystal chitin structures with catalytically modifying participation of gene expression products.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters., Results: Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs., Conclusions: The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other species, O. pardalina, we found no clear trace of concerted evolution for the dispersed hsp70 genes. Furthermore, in the latter species we detected hsp70 pseudogenes, representing a hallmark of the birth-and-death process.

The protein pattern of mouse macrophage strain (J774) has been investigated using 2D electrophoresis after combined action of bacterial endotoxins (LPS), heat shock treatment (HS) and administration of recombinant human Hsp70. The investigation demonstrated significant protective effect of HS and recombinant Hsp70 treatment applied before LPS introduction. This effect is apparently realized by means of several signal transduction systems. In the course of the investigation, we have identified eight proteins, which exhibited pronounced changes in their synthesis due to combined treatment. The data accumulated may shed light on molecular mechanisms underlying protective antiseptic action of HS and/or recombinant Hsp70 applied before LPS administration.

Heat shock protein 70 (Hsp70) is a molecular chaperone providing tolerance to heat and other challenges at the cellular and organismal levels. We sequenced a genomic cluster containing three hsp70 family genes linked with major histocompatibility complex (MHC) class III region from an extremely heat tolerant animal, camel (Camelus dromedarius). Two hsp70 family genes comprising the cluster contain heat shock elements (HSEs), while the third gene lacks HSEs and should not be induced by heat shock. Comparison of the camel hsp70 cluster with the corresponding regions from several mammalian species revealed similar organization of genes forming the cluster. Specifically, the two heat inducible hsp70 genes are arranged in tandem, while the third constitutively expressed hsp70 family member is present in inverted orientation. Comparison of regulatory regions of hsp70 genes from camel and other mammals demonstrates that transcription factor matches with highest significance are located in the highly conserved 250-bp upstream region and correspond to HSEs followed by NF-Y and Sp1 binding sites. The high degree of sequence conservation leaves little room for putative camel-specific regulatory elements. Surprisingly, RT-PCR and 5'/3'-RACE analysis demonstrated that all three hsp70 genes are expressed in camel's muscle and blood cells not only after heat shock, but under normal physiological conditions as well, and may account for tolerance of camel cells to extreme environmental conditions. A high degree of evolutionary conservation observed for the hsp70 cluster always linked with MHC locus in mammals suggests an important role of such organization for coordinated functioning of these vital genes.

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The influence of the P-element built into the area of the CG5017 gene on the mutation of the spineless (ss) gene was studied. It was shown that the insertion of the P-element decreased the level of transcription of CG5017 approximately twofold. Modulation of the level of transcription of the CG5017 gene helped demonstrate, for the first time, its influence on the phenotypic manifestation of the mutation of the ss gene, which shows their interaction in the process of regulation of morphogenesis of limbs in Drosophila melanogaster.

The tissue-specific accumulation of small bristles (Dm nxf1) transcripts at different developmental stages of Drosophila melanogaster was analyzed by Northern blots and RT PCR. We identified four distinct transcripts: ubiquitous (3.5kb); ovary and early embryo specific (3.3kb); testis specific (1.9kb and 2.8kb) and nervous system specific (5.1kb). The pattern of Dm nxf1 gene expression in ovaries and early embryos (0-2h) is similar: the sizes of transcripts range from 3.0 to 3.5kb. We propose that this size variability may reflect the different extent of cytoplasmic polyadenylation. In testes, the 2.8-kb transcript originates from alternative termination of transcription and the 1.9-kb transcript is supposed to originate from an alternative transcription start. During ontogenesis, the 5.1-kb transcript can be clearly detected in 10- to 18-h-old embryos, most prominently in the nervous ganglia of larvae, and it represents a major species in imago head extracts. We found that the 5.1-kb transcript, similarly to the nxf1 heavy transcripts in Homo sapiens and Mus musculus, results from the retention of intron 5-6 that corresponds to the intron 10-11 in Hs nxf1 and Mm nxf1 genes., (Copyright 2010 Elsevier B.V. All rights reserved.)

Colon cancer is one of the leading causes of cancer deaths in developed countries due to the absence of tumor specific markers for early diagnosis of the disease, providing adequate sensitivity. Search for diagnostic markers of various types of cancer by proteomic approaches has been limited by large differences in protein centration. We used preliminary extraction of major cellular proteins by 0.2 M sodium chloride in presence of nonionic detergent NP-40 in order to raise the sensitivity of the 2D PAGE detection of low-abundant soluble proteins, some of which may penetrate in blood circulation during carcinogenesis. Application of this procedure prior to 2D comparative analysis of proteomes of normal tissues and matched colon cancer specimens led to selection of ten proteins, which are frequently overexpressed in colon adenocarcinomas. Mass-spectrometric identification of selected proteins led to discovery of two novel protein markers of colon tumors--TAF9 and CISH. Low level of CISH expression in various tissues suggests that it is a novel prospective marker for diagnosis of colon cancer.

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumor in comparison to normal tissues. Despite the wide application of proteomics in current studies, identification of proteins with stable concentration differences in normal and cancer cells remains rather difficult. The current study was directed to the search of new potential protein colorectal cancer markers using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. This widespread neoplasm is characterized by lack of evident symptoms at early stages of cancerogenesis. It is highly important to develop fast and sensitive methods of molecular diagnostics. We studied paired cancerous and normal clinical tissue samples from 11 patients with colorectal adenocarcinomas by comparative 2-D PAGE and MALDI-TOF mass-spectrometry identification. Sixteen proteins with stable differential expression were selected and identified, including 13 overexpressed and 3 downregulated proteins. In summary, we describe the discovery overexpression of GPD1 and RRBP1 proteins and lack of expression for HNRNPH1 and SERPINB6 proteins which are new candidate biomarkers of colon cancer.

We cloned and sequenced one of Drosophila montana hsp70 genes. 3'-flanking region of this particular copy contains fragment of SGM mobile element. Previously this element was found within hsp70 3'-flanking region of other species of the virilis species group namely D. virilis and D. lummei. We have described reorganization of hsp70 gene cluster in one of D. virilis strains involving full-length SGM. Our data enable one to suggest evolutionary conservatism of SGM localization within hsp70 gene cluster of different species of the virilis group of Drosophila and implicate this mobile element in the evolution of hsp70 genes.

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name 'soldier flies', occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 degrees C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.

We compared a series of Drosophila strains with P element insertions from -28 to -144 nucleotides 5' to the transcription start site of the Hsp70A genes-corresponding to the range of naturally occurring P element insertion sites-to elucidate the consequences of insertion site for Hsp70A gene expression. Although all insertions reduced Hsp70A expression below that of a control strain, the magnitude of the reduction was inversely related to the number of nucleotides between the transcription start site and the insertion site. A pre-existing hypothesis is that naturally occurring transposable element insertions in Hsp promoters may be beneficial in some circumstances, which may account for their retention in natural populations. In the present study, in a control line heat shock reduced fecundity, whereas in lines with P element insertions heat shock typically increased fecundity. Finally, according to cluster-specific quantitative RT-PCR, expression of the Hsp70A cluster genes was typically greater than that of the Hsp70B gene cluster genes, although the latter are more numerous and, in this case, free of P element insertions.

The hsp70 genes is among the main systems underlying the adaptation of organisms to adverse environmental factors. The ever increasing amount of data in literature demonstrates an important adaptive role of mobile genetic elements in microevolution. Drosophila hsp70 genes are potential target for transpositions of various mobile elements in natural populations. We have analyzed the frequency and localization of a P element-based genetic construction, EPgy2, in the region of Drosophila melanogaster hsp70 genes. A hot spot for the transposition was discovered in the promoter regions of genes hsp70Aa and hsp70Ab. No insertions of this construction in the coding or 3'-flanking regions of hsp70 genes have been recorded. It was demonstrated that the region of 161 to 7800 bp adjacent to the original construction is in certain cases also involved in the transposition. No transpositions of any other mobile elements have been observed. The inserts were shown to change the activity of hsp70 genes and the thermotolerance of transgenic strains.

For many years,we and our collaborators have investigated the adaptive role of heat shock proteins in different animals,including the representatives of homothermic and poikilothermic species that inhabit regions with contrasting thermal conditions. Adaptive evolution of the response to hyperthermia has led to different results depending upon the species. The thermal threshold of induction of heat shock proteins in desert thermophylic species is, as a rule, higher than in the species from less extreme climates. In addition,thermoresistant poikilothermic species often exhibit a certain level of heat shock proteins in cells even at a physiologically normal temperature. Furthermore,there is often a positive correlation between the characteristic temperature of the ecological niche of a given species and the amount of Hsp70-like proteins in the cells at normal temperature. Although in most cases adaptation to hyperthermia occurs without changes in the number of heat shock genes, these genes can be amplified in some xeric species. It was shown that mobile genetic elements may play an important role in the evolution and fine-tuning of the heat shock response system,and can be used for direct introduction of mutations in the promoter regions of these genes.