Alterations of serum biochemical parameters and tyrosine phosphorylation in kidney and liver of chronic stress-induced rats

Abstract Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male SpragueDawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.

Resumo O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.

Alterations of serum biochemical parameters and tyrosine phosphorylation in kidney and liver of chronic stress-induced rats / Alterações dos parâmetros bioquímicos séricos e fosforilação da tirosina em rim e fígado de ratos induzidos por estresse crônico

Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male Sprague-Dawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.

O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.

Identification of Leishmania species by next generation sequencing of hsp70 gene.

Leishmaniasis is a major public health problem worldwide. Although next generation sequencing technology has been widely used in the diagnosis of infectious diseases, it has been scarcely applied in identification of Leishmania species. The aim of this study was to compare the efficiency of MinION™ nanopore sequencing and polymerase chain reaction restriction fragment length polymorphism in identifying Leishmania species. Our results showed that the MinION™ sequencer was able to discriminate reference strains and clinical samples with high sensitivity in a cost and time effective manner without the prior need for culture.

Molecular investigations of the effect of thermal manipulation during embryogenesis on muscle heat shock protein 70 and thermotolerance in broiler chickens

The objective of this study was to elucidate the optimum protocol timing of thermal manipulation (TM) during embryogenesis, which underline genetic improvement of muscle thermotolerance acquisition. For the present study, 1,440 fertile eggs were divided randomly and equally into control (37.8 °C with 56% relative humidity) and four thermally manipulated groups (TM1, TM2, TM3, and TM4) subjected to 39 °C for 18 h with 65% relative humidity daily during different embryonic periods. Then, at day 35 post-hatch, all groups were subjected to thermal challenge at 43 °C for 6 h to identify the level of thermotolerance acquisition differences between them. Hsp70 mRNA expression was evaluated by using a relative quantitatively RT-qPCR. Single nucleotide polymorphisms sequence of the Hsp70 gene was evaluated by Sanger's sequencing method. Pectoral and thigh muscles samples were subjected to immunohistochemistry to detect Hsp70. Among TM conditions that were investigated, TM1 (39 °C for 18 h during embryonic days (ED) 7­11) induced a significant improvement in thermotolerance parameters (body temperature and T3 levels) during thermal challenge combined with an increase in the levels of Hsp70 mRNA and its protein with a high stability of nucleotide sequences in both pectoral and thigh muscles. The partial DNA sequence of Hsp70 gene in TM1 was reported, and nucleotide sequences were deposited in NCBI GenBank database with the accession numbers (MK852579) and (MK852580). Thigh muscle thermotolerance acquisition was higher than pectoral muscle during thermal challenge at 43 °C for 6 h. Thus, TM during ED7­11 may improve thermotolerance acquisition without adversely affecting performance.(AU)

Antigiardial Activity of Acetylsalicylic Acid Is Associated with Overexpression of HSP70 and Membrane Transporters.

Giardia lamblia is a flagellated protozoan responsible for giardiasis, a worldwide diarrheal disease. The adverse effects of the pharmacological treatments and the appearance of drug resistance have increased the rate of therapeutic failures. In the search for alternative therapeutics, drug repositioning has become a popular strategy. Acetylsalicylic acid (ASA) exhibits diverse biological activities through multiple mechanisms. However, the full spectrum of its activities is incompletely understood. In this study we show that ASA displayed direct antigiardial activity and affected the adhesion and growth of trophozoites in a time-dose-dependent manner. Electron microscopy images revealed remarkable morphological alterations in the membrane, ventral disk, and caudal region. Using mass spectrometry and real-time quantitative reverse transcription (qRT-PCR), we identified that ASA induced the overexpression of heat shock protein 70 (HSP70). ASA also showed a significant increase of five ATP-binding cassette (ABC) transporters (giABC, giABCP, giMDRP, giMRPL and giMDRAP1). Additionally, we found low toxicity on Caco-2 cells. Taken together, these results suggest an important role of HSPs and ABC drug transporters in contributing to stress tolerance and protecting cells from ASA-induced stress.

Detailed Characterization of the Cooperative Binding of Piperine with Heat Shock Protein 70 by Molecular Biophysical Approaches.

In this work, for the first time, details of the complex formed by heat shock protein 70 (HSP70) independent nucleotide binding domain (NBD) and piperine were characterized through experimental and computational molecular biophysical methods. Fluorescence spectroscopy results revealed positive cooperativity between the two binding sites. Circular dichroism identified secondary conformational changes. Molecular dynamics along with molecular mechanics Poisson Boltzmann surface area (MM/PBSA) reinforced the positive cooperativity, showing that the affinity of piperine for NBD increased when piperine occupied both binding sites instead of one. The spontaneity of the complexation was demonstrated through the Gibbs free energy (∆G < 0 kJ/mol) for different temperatures obtained experimentally by van't Hoff analysis and computationally by umbrella sampling with the potential of mean force profile. Furthermore, the mean forces which drove the complexation were disclosed by van't Hoff and MM/PBSA as being the non-specific interactions. In conclusion, the work revealed characteristics of NBD and piperine interaction, which may support further drug discover studies.

Expression of Heat shock protein 70 (Hsp70-1) in Plasmodium berghei ookinetes and its participation in midgut mosquito infection.

The heat shock protein family 70 (Hsp70) comprises chaperone proteins that play major multiple roles in Plasmodium asexual and sexual development. In this study, we analyzed the expression of Hsp70-1 in gametocytes, gametes, zygotes, and its participation in ookinete formation and their transition into oocysts. A monoclonal antibody against recombinant Hsp70-1 revealed its presence in zygotes and micronemes of ookinetes. Compared to wild type parasites, Hsp70-1 knockout ookinetes produced fewer oocysts in Plasmodium-susceptible Anopheles albimanus mosquitoes. This may indicate a defective transformation of ookinetes into oocysts in the absence of Hsp70-1. The presence of this protein in micronemes suggests its participation in mosquito infection, probably aiding to the adequate structural conformation of proteins in charge of motility, recognition and invasion of the insect midgut epithelium.

Alterations on a key nephrogenic/cardiogenic gene expression linked to hypertension development.

The elevation of blood pressure produces specific organic lesions, including kidney and cardiac damage. On the other hand, cardiovascular disease usually leads to the development of hypertension. Thus, hypertension could be both a cause and a consequence of cardiovascular disease. Previous studies linked the lack of nitric oxide to cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced endothelium-derived hyperpolarizing factor responses, with shorter survival. The lack of this gas also leads to renal/cardiac abnormalities. It is widely known that nephrogenic deficiency is a risk factor for kidney disease. Besides, recent evidence suggests that alterations in WT-1, a key nephrogenic factor, could contribute to the development of hypertension. Moreover, some genes involved in the development of hypertension depend on WT-1. This knowledge makes it essential to investigate and understand the mechanisms regulating the expression of these genes during renal/cardiac development, and hypertension. As a consequence, the most in-depth knowledge of the complex aetiopathogenic mechanism responsible for the hypertensive disease will allow us to propose novel therapeutic tools.

Implications of the transcription factor WT1 linked to the pathologic cardiac remodeling post-myocardial infarction. / Implicaciones del factor de transcripción WT1 asociado al remodelado cardiaco patológico postinfarto miocárdico.

New advances in the treatment of acute myocardial infarction involve novel signaling pathways and cellular progeny. In this sense, regeneration is a novel tool that would contribute to post-infarction physiological ventricular remodeling. More specifically, re-expression of the WT1 transcription factor in the myocardial wall by ischemia and infarction would be related to the invasion of cells with the capacity for regeneration. This mechanism seems not to be sufficient to restore muscle cells and lost vessels entirely. Of particular interest, the presence of the heat-shock response protein 70 (Hsp70) and its interaction with the vitamin D receptor would modulate the expression of WT1 positively. In this context, it is proposed that the activation of vitamin D receptors associated with Hsp70 could favor physiological cardiac remodeling and reduce the progression to heart failure.

Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs.

A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.

Molecular epidemiology of Babesia vogeli in dogs from the southeastern region of Rio de Janeiro, Brazil.

Hemoparasitic diseases are prominent in domestic animals, particularly in Brazil, a tropical country with a wide range of vectors. This study investigated the epidemiology of Babesia vogeli in the whole blood of dogs from the southeastern region of Rio de Janeiro, Brazil. Whole blood samples from 390 dogs were screened for the presence of B. vogeli DNA by qPCR using the heat shock protein 70 kDa (hsp70) gene of B. vogeli. Characteristics related to the host and its environment were collected using a questionnaire. Bivariate analysis was used to evaluate each factor individually. A phi correlation test was used to verify collinearity. The variables with p < .1 and a low or moderate correlation with the other variables were selected for the multivariate analysis. Multiple models were created, and the best logistic regression model was chosen using the Akaike Information Criterion (AIC). The final model was used to determine which variables were closely related to B. vogeli infections in dogs. Of the 390 dog blood samples, 15.66% were positive for B. vogeli. The variables cat contact, age, shelter, street or woods access, tick infestation and fur lengthwere included in the final model. Per the logistic regression analysis, three variables explained B. vogeli detection in dogs: age (odds ratio [OR] = 2.12; p-value <.05; confidence interval [CI]: 1.13-3.96), tick infestation (OR = 2.08; p-value <.05; CI: 1.10-3.93) and shelter (OR = 2.22; p-value <.05; CI: 1.16-4.26). These variables were determined to be associated with B. vogeli detection in domiciled dogs in the southeastern region of Rio de Janeiro, Brazil. These data indicate that the age of the animal, the presence of ticks and the lack of shelter directly affect the epidemiology of B. vogeli.

Putative model for heat shock protein 70 complexation with receptor of advanced glycation end products through fluorescence proximity assays and normal mode analyses.

Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.

Growing evidence suggests WT1 effects in the kidney development are modulated by Hsp70/NO interaction.

The study of kidney development at the cellular and molecular levels remains an active area of nephrology research. The functional integrity of the kidney depends on normal development as well as on physiological cell turnover. Apoptosis induction is essential for these mechanisms. A route to cell death revealed in the past decade shows that heat shock proteins (HSPs) and their cofactors are responsible for regulating the apoptotic pathway. Specifically, heat shock protein 70 (Hsp70), the most ubiquitous and highly conserved HSP, helps proteins adopt native conformation or regain function after misfolding. Hsp70 is an important cofactor for the function of Wilms' tumour 1 (WT1) and suggests a potential role for this chaperone during kidney differentiation. In addition, we have demonstrated that WT1 expression is modulated by nitric oxide (NO) availability and Hsp70 interaction after neonatal unilateral ureteral obstruction. NO has been identified as playing an important role in the developing kidney. These findings suggest that Hsp70 and NO may play a critical and fundamental role in the capacity to modulate both apoptotic pathway and oxidative stress during kidney development. Furthermore, the design of experimental protocols that assess renal epithelial functionality in this context, could contribute to the understanding of renal development and alterations.

Changes in renal WT-1 expression preceding hypertension development.

BACKGROUND: Hypertension is a public health problem with mostly unknown causes, and where strong hereditary genetic alterations have not been fully elucidated. However, the use of experimental models has provided valuable information. Recent evidences suggest that alterations in key nephrogenic factors, such as Wilms' tumor 1 transcription factor (WT-1), could contribute to the development of hypertension. The aim of this paper is to evaluate the expression of WT-1 and related genes in the nephrogenic process in connection with the development of hypertension as well as the corresponding anatomical and functional correlation. METHODS: Male spontaneously hypertensive and control rats were evaluated weekly from birth until week 8 of life. Their blood pressure was taken weekly using the tail-cuff blood pressure system. Weekly, 5 rats per group were sacrificed with a lethal injection of pentobarbital, and their kidneys were removed, decapsulated and weighed. The serum was collected for measuring biochemical parameters. The results were assessed using one-way analysis of variance for comparisons between groups. RESULTS: The relationship between renal weight/total body weights was established, without significantly different values. These data were compared with apoptosis, fibrosis, number and size of the glomeruli. The elevation of systolic blood pressure was significant since week 6. Biochemical values differed slightly. Histology showed a slight increase in deposits of collagen fibers since week 4. Additionally, in kidney cortices, the expression of WT-1, heat shock protein 70 (Hsp70) and vitamin D receptors (VDR) decreased since week 4. Finally, we demonstrated ultrastructural damage to mitochondria since week 4. CONCLUSIONS: Our results would suggest an unprecedented link, possibly a regulatory mechanism, between WT-1 on nephrogenic alteration processes and their relationship with hypertension. Moreover, and previous to the increase in blood pressure, we demonstrated low expressions of WT-1, VDR and Hsp70 in kidneys from neonatal SHRs. If so, this may suggest that deregulation in the expression of WT-1 and its impact on nephrogenesis induction could be crucial in understanding the development and maintenance of hypertension.

Emerging Role of Nitric Oxide and Heat Shock Proteins in Insulin Resistance.

Insulin resistance (IR) is present in pathologies such as diabetes, obesity, metabolic syndrome, impaired glucose tolerance, hypertension, inflammation, cardiac disease, and dyslipidemias. Population studies show that IR is multifactorial and has genetic components, such as defects in the insulin-signaling pathway (as serine phosphorylation on insulin substrate or decreased activation of signaling molecules) and RAS/MAPK-dependent pathways. IR is connected to mitochondrial dysfunction, overproduction of oxidants, accumulation of fat, and an over-activation of the renin-angiotensin system linked to the NADPH oxidase activity. In addition, nitric oxide (NO), synthesized by nitric oxide synthases (endothelial and inducible), is also associated with IR when both impaired release and reduced bioavailability of all which lead to inflammation and hypertension. However, increased NO may promote vasculoprotection. Moreover, reduced NO release induces heat shock protein 70 kDa (HSP70) expression in IR and diabetes, mediating beneficial effects against oxidative stress injury, inflammation and apoptosis. HSP70 may be used as biomarker of the chronicity of diabetes. Hsp72 (inducible protein) is linked to vascular complications with a high-fat diet by blocking inflammation signaling (cytoprotective and anti-cytotoxicity intracellular role). Elucidating the IR signaling pathways and the roles of NO and HSPs is relevant to the application of new treatments, such as heat shock and thermal therapy, nitrosylated drugs, chemical chaperones or exercise training.

Mediators and mechanisms of heat shock protein 70 based cytoprotection in obstructive nephropathy.

Urinary heat shock protein 70 (Hsp70) is rapidly increased in patients with clinical acute kidney injury, indicating that it constitutes a component of the endogenous stress response to renal injury. Moreover, experimental models have demonstrated that Hsp70 activation is associated with the cytoprotective actions of several drugs following obstruction, including nitric oxide (NO) donors, geranylgeranylacetone, vitamin D, and rosuvastatin. Discrete and synergistic effects of the biological activities of Hsp70 may explain its cytoprotective role in obstructive nephropathy. Basic studies point to a combination of effects including inhibition of apoptosis and inflammation, repair of damaged proteins, prevention of unfolded protein aggregation, targeting of damaged protein for degradation, and cytoskeletal stabilization as primary effectors of Hsp70 action. This review summarizes our understanding of how the biological actions of Hsp70 may affect renal cytoprotection in the context of obstructive injury. The potential of Hsp70 to be of central importance to the mechanism of action of various drugs that modify the genesis of experimental obstructive nephropathy is considered.

The relevance of dengue virus genotypes surveillance at country level before vaccine approval.

Dengue is a major threat for public health in tropical and subtropical countries around the world. In the absence of a licensed vaccine and effective antiviral therapies, control measures have been based on education activities and vector elimination. Current efforts for developing a vaccine are both promising and troubling. At the advent of the introduction of a tetravalent dengue vaccine, molecular surveillance of the circulating genotypes in different geographical regions has gained considerable importance. A growing body of in vitro, preclinical, and clinical phase studies suggest that vaccine conferred protection in a geographical area could depends on the coincidence of the dengue virus genotypes included in the vaccine and those circulating. In this review we present the state-of-the-art in this field, highlighting the need of deeper knowledge on neutralizing immune response for making decisions about future vaccine approval and the potential need for different vaccine composition for regional administration.

The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells.

The pathogenic yeast Cryptococcus neoformans secretes numerous proteins, such as heat shock proteins, by unconventional mechanisms during its interaction with host cells. Hsp70 is a conserved chaperone that plays important roles in various cellular processes, including the interaction of fungi with host immune cells. Here, we report that sera from individuals with cryptococcosis infection recognize a recombinant C. neoformans Hsp70 (Cn_rHsp70). Moreover, immunofluorescence assays using antibodies against Cn_rHsp70 revealed the localization of this protein at the cell surface mainly in association with the capsular network. We found that the addition of Cn_rHsp70 positively modulated the interaction of C. neoformans with human alveolar epithelial cells and decreased fungal killing by mouse macrophages, without affecting phagocytosis rates. Immunofluorescence analysis showed that there was a competitive association among the receptor, GXM and Cn_rHsp70, indicating that the Hsp70-binding sites in host cells appear to be shared by glucuronoxylomannan (GXM), the major capsular antigen in C. neoformans. Our observations suggest additional mechanisms by which Hsp70 influences the interaction of C. neoformans with host cells.

Effect of 60 Hz electromagnetic fields on the activity of hsp70 promoter: an in vivo study.

Exposure to EMFs (electromagnetic fields) results in a number of important biological changes, including modification of genetic expression. We have investigated the effect of 60 Hz sinusoidal EMFs at a magnetic flux density of 80 µT on the expression of the luciferase gene contained in a plasmid labelled as pEMF (EMF plasmid). This gene construct contains the specific sequences for the induction of hsp70 (heat-shock protein 70) expression by EMFs, as well as the reporter for the luciferase gene. The pEMF vector was electrotransferred into quadriceps muscles of BALB/c mice that were later exposed to EMFs. Increased luciferase expression was observed in mice exposed to EMFs 2 h daily for 7 days compared with controls (P<0.05). These data along with other reports in the literature suggest that EMFs can have far-reaching effects on the genome.

Expressão de proteínas de choque térmico na epilepsia do lobo temporal mesial / Heat shock protein expression in mesial temporal lobe epilepsy

OBJETIVOS: Em modelos animais de epilepsia, heat shock protein 70 (HSP70) tem sua expressão proporcional à intensidade de crises. A HSP90, dentre diversas ações, regula a sintase neuronal do óxido nítrico e proteínas do citoesqueleto. Devido ao provável papel protetor de HSP70 e à relação de HSP90 com proteínas envolvidas na epileptogênese, decidimos investigar a expressão imunohistoquímica destas proteínas na epilepsia do lobo temporal mesial (ELTM). MÉTODOS: Hipocampos de pacientes ELTM fármaco-resistentes foram obtidos durante o procedimento cirúrgico e hipocampos controle foram obtidos de necrópsias. Os espécimes obtidos foram tratados igualmente e submetidos a imunohistoquímica. Medidas de imuno-reatividade positiva foram obtidas com o software ImageJ. RESULTADOS: Nossas medidas mostraram menor expressão de HSP70 e HSP90 no hipocampo de pacientes epilépticos do que nos controles em praticamente todas as regiões do hipocampo. Para HSP70 as diferenças significativas foram encontradas na região subicular e para HSP90 em todas, exceto fascia dentata e subículo. CONCLUSÃO: Diferente dos achados em modelos animais, nossos resultados indicam que crises crônicas nos pacientes ELTM não são estímulo suficiente para ativação exacerbada de HSP70 e HSP90. Condições inerentes à ELTM podem ser determinantes desta menor expressão. Ainda, nossos achados sugerem que a baixa expressão de HSPs pode estar relacionada a manutenção das crises.

OBJECTIVE: In animal models of epilepsy, heat shock protein 70 (HSP70) has its expression proportional to seizure severity. Among several functions on biological systems, HSP90 regulates nitric oxide synthase and cytoskeletal proteins. Due to the plausible protective role of HSP70 and the relationship of HSP90 with proteins involved in epileptogenesis, we looked at HSP70 and 90 immunohistochemical expression in temporal lobe epilepsy (TLE). METHODS: Hippocampi were obtained from medically intractable TLE patients and control hippocampi were from necropsy cases. Specimens were equally treated and submitted to imunohistochemistry to HSP70 and HSP90. Positive immunoreactivity was estimated using the software ImageJ. RESULTS: Our results showed significant lower expression of HSP70 and HSP90 in epileptic patients when compared to controls in almost all hippocampal regions. To HSP70 subicular region exhibited significant difference and to HSP90 all regions, except fascia dentata and subiculum. CONCLUSION: Unlike the reports in animal models the present results indicate that chronic seizures in TLE patients are not sufficient to induce HSP70 and HSP90 activation. Typical attributes inherent to TLE condition may be determinants of low HSP expression. In Addition, our results suggest that low expression of HSPs in epileptic groups may be related to seizure maintenance.