Caracterização do gene do choque térmico (HSP-70. 1) e sua relação com características de produção em bovinos leiteiros criados no semiárido brasileiro / Characterization of the thermal shock gene (HSP-70. 1) and its relationship with production characteristics in dairy cattle reared in the Brazilian semiarid

Objetivou-se com este trabalho avaliar a diversidade genética do gene HSP-70.1 e associar os polimorfismos encontrados com a performance de vacas leiteiras das raças Holandesa, Girolando (5/8H-G) e Sindi criadas em região do semiárido brasileiro. Os polimorfismos foram identificados e avaliados pela técnica de PCR-RFLP, usando-se a enzima de restrição EcoRII. Avaliou-se a variabilidade genética por meio do índice de diversidade padrão e da análise de variância molecular (AMOVA). Os polimorfismos identificados foram avaliados sobre as características de produção de leite. Foram identificados sete alelos, os quais demonstraram que houve polimorfismo para a região gênica analisada, e alguns alelos foram compartilhados entre os rebanhos. As raças bovinas Holandesa e Sindi foram similares geneticamente para o gene analisado. A AMOVA demonstrou que há variação genética entre os rebanhos e dentro deles, com a maior parte da variação ocorrendo dentro dos rebanhos para todos os grupos avaliados. Houve efeito dos alelos identificados sobre a produção de leite dos rebanhos das raças Holandesa (P<0,0001) e Girolando (P<0,0117). O gene HSP-70.1 foi polimórfico na população de bovinos leiteiros estudada, sendo, portanto, um marcador molecular promissor para avaliar a produção de leite de raças criadas em região semiárida.(AU)

The objective of this work was to evaluate the genetic diversity of the HSP-70.1 gene and to associate the polymorphisms found with the performance of Holstein, Girolando (5/8H-G) and Sindi dairy cows raised in region of the Brazilian semiarid. Polymorphisms were identified and evaluated using the PCR-RFLP technique using the EcoRII restriction enzyme. Genetic variability was evaluated using the standard diversity index and molecular variance analysis (AMOVA). The identified polymorphisms were evaluated on the characteristics of milk production. They were identified from the seven alleles, demonstrating that there was polymorphism for the analyzed gene region and some alleles were shared among the herds. The Holstein and Sindi bovine breeds were genetically like the analyzed gene. AMOVA demonstrated that there is genetic variation between and within the herds, with most of the variation occurring within the herds for all groups evaluated. There was effect of the alleles identified on the production of milk herds of Holstein and (P<0.0001) Girolando (P<0.0117) breeds. The HSP-70.1 gene was polymorphic in the population of dairy cattle studied, and therefore a promising molecular marker to evaluate milk production of breeds created in semiarid regions.(AU)

Mucosal leishmaniasis: A forgotten disease, description and identification of species in 50 Colombian cases / Leishmaniasis mucosa: una enfermedad olvidada, descripción e identificación de especies en 50 casos colombianos

Abstract Introduction: Mucosal leishmaniasis has a progressive course and can cause deformity and even mutilation in the affected areas. It is endemic in the American continent and it is mainly caused by Leishmania (Viannia) braziliensis. Objective: To describe a series of mucosal leishmaniasis cases and the infectious Leishmania species. Materials and methods: We included 50 patients with a clinical diagnosis of mucosal leishmaniasis and parasitological confirmation, and we described their clinical and laboratory results. We performed species typing by PCR-RFLP using the miniexon sequence and hsp70 genes; confirmation was done by sequencing. Results: The median time of disease evolution was 2.9 years (range: 1 month to 16 years). The relevant clinical findings included mucosal infiltration (94%), cutaneous leishmaniasis scar (74%), total loss of the nasal septum (24%), nasal deformity (22%), and mucosal ulceration (38%). The symptoms reported included nasal obstruction (90%), epistaxis (72%), rhinorrhea (72%), dysphonia (28%), dysphagia (18%), and nasal pruritus (34%). The histopathological study revealed a pattern compatible with leishmaniasis in 86% of the biopsies, and amastigotes were identified in 14% of them. The Montenegro skin test was positive in 86% of patients, immunofluorescence in 84%, and culture in 8%. Leishmania (V.) braziliensis was identified in 88% of the samples, L. (V) panamensis in 8%, and L. (V.) guyanensis and L. (L.) amazonensis in 2% respectively. Conclusion: In this study, we found a severe nasal disease with destruction and deformity of the nasal septum in 25% of the cases, probably associated with late diagnosis. Leishmania (V.) braziliensis was the predominant species. We described a case of mucosal leishmaniasis in Colombia caused by L. (L.) amazonensis for the first time.

Resumen Introducción. La leishmaniasis mucosa tiene un curso progresivo y puede causar deformidad e incluso mutilación de las zonas afectadas. Es endémica en el continente americano y es causada principalmente por Leishmania (Viannia) brasiliensis. Objetivo. Describir una serie de casos de leishmaniasis mucosa y las especies de Leishmania infecciosas. Materiales y métodos. Se estudiaron 50 pacientes con diagnóstico clínico de leishmaniasis mucosa y confirmación parasitológica. Se describieron sus características clínicas y los resultados de laboratorio. La tipificación de especies se hizo mediante reacción en cadena de la polimerasa de los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism Polymerase Chain Reaction, PCR-RFLP) en la secuencia del miniexon y el gen hsp70 y se confirmó por secuenciación. Resultados. La evolución de la enfermedad fue de un mes a dieciséis años (mediana de 2,8 años). Los hallazgos clínicos fueron los siguientes: infiltración mucosa (94 %), cicatriz de leishmaniasis cutánea (74 %), pérdida total del tabique nasal (24 %), deformidad nasal (22 %) y ulceración (38 %). Los síntomas reportados fueron: obstrucción nasal (90 %), epistaxis (72 %), rinorrea (72 %), disfonía (28 %), disfagia (18 %) y prurito nasal (34 %). La histopatología mostró un patrón compatible con leishmaniasis en 86 % de las biopsias y se identificaron amastigotes en 14 % de ellas. La prueba de Montenegro fue positiva en 86 % de los pacientes, la inmunofluorescencia en 84 %, y el cultivo en 8 %. Leishmania (V.) brasiliensis se identificó en 88 % de las muestras, L. (V) panamensis en 8 %, y L. (V.) guyanensis y L. (L.) amazonensis en 2 %, respectivamente. Conclusión. Se encontró enfermedad nasal grave con destrucción y deformidad del tabique nasal en una cuarta parte de los casos, probablemente debido a un diagnóstico tardío. Leishmania (V.) brasiliensis fue la especie predominante. Se describe por primera vez un caso de leishmaniasis mucosa causado por L. (L.) amazonensis en Colombia.

Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia / Vigilancia de la fauna flebotomínea e infección de Didelphis marsupialis (Didelphimorphia: Didelphidae) en un área de alta endemia de leishmaniasis visceral en Colombia

Abstract Introduction: The study of the interaction between the parasite, the vector and the mammalian hosts, including man, allows to understand the behavior of the leishmaniases. Objective: To determine the presence of Lutzomyia species and to detect the Leishmania infection in Didelphis marsupialis in an endemic area for visceral leishmaniasis. Materials and methods: Phlebotomine fauna and individuals of D. marsupialis were collected with CDC and Tomahawk™ traps, respectively. The species of Lutzomyia were identified using the Young and Duncan key (1994). Ear and tail biopsies and blood samples from D. marsupialis were taken to identify the Leishmania species by amplifying a fragment of the gene associated with the 70 kD heat shock protein. Results: Seven Lutzomyia species were identified: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana and Lu. trinidadensis. The first three species have epidemiological importance in Colombia because of their implications in the transmission of the Leishmania parasite. Sixty-five tissue samples from 19 D. marsupialis individuals were negative for Leishmania spp. Conclusions: The presence of the Lutzomyia species that have been identified as vectors for Leishmania inside and around houses in the village of El Bledo, in El Carmen de Bolívar represents a risk of infection. Furthermore, the presence of Lu. panamensis is reported for first time in El Carmen de Bolívar in Colombia. Although the lack of detection of Leishmania spp. in D. marsupialis samples may suggest that D. marsupialis does not play an important role in the transmission cycle of Leishmania in this region, it is necessary to carry out further longitudinal studies to confirm this hypothesis.

Resumen Introducción. El estudio de la interacción entre el parásito, el vector y los huéspedes mamíferos, incluido el hombre, permite entender el comportamiento de la leishmaniasis. Objetivo. Determinar la presencia de especies del género Lutzomyia y detectar la infección por Leishmania spp. en Didelphis marsupialis en un área endémica de leishmaniasis visceral. Materiales y métodos. Se recolectaron flebotomíneos y D. marsupialis con trampas CDC y Tomahawk™, respectivamente. Las especies de Lutzomyia se identificaron usando la clave de Young y Duncan, 1994. Se tomaron biopsias de oreja, cola y muestras de sangre de D. marsupialis para diagnosticar Leishmania spp. mediante la amplificación de un fragmento del gen de la proteína de choque térmico de 70 kD. Resultados. Se identificaron siete especies de Lutzomyia: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana y Lu. trinidadensis. Las tres primeras especies son reconocidas como vectores en el país por estar implicadas en la transmisión de Leishmania spp. En total, 65 muestras de tejidos de oreja, cola y de sangre provenientes de 19 D. marsupialis fueron negativas para Leishmania spp. en la PCR-HSP70. Conclusiones. La presencia de flebotomíneos con importancia epidemiológica en la zona evaluada representa un riesgo de transmisión. Asimismo, Lu. panamensis es reportada por primera vez en El Bledo (Carmen de Bolívar). La ausencia de Leishmania spp. en D. marsupialis podría sugerir que esta especie no tiene un papel importante en el ciclo de transmisión de Leishmania en la vereda El Bledo, por lo que es necesario profundizar en estudios longitudinales para corroborar esta hipótesis.

Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study / Tipificación molecular de Leishmania (Leishmania) amazonensis y especies del subgénero Viannia asociadas a la leishmaniasis cutánea y la mucosa en Colombia: un estudio de concordancia

Resumen Introduction: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. Objective: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. Materials and methods: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. Results: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Conclusion: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Abstract Introducción. La electroforesis de enzimas multilocus (Multilocus Enzyme Electrophoresis, MLEE) es el estándar de referencia para la tipificación de las especies de Leishmania. La prueba está restringida a laboratorios especializados por su complejidad técnica, sus costos y el tiempo necesario para obtener resultados. La PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) se utiliza para tipificar especies de Leishmania. Objetivo. Establecer la concordancia entre las dos pruebas como métodos de tipificación de las especies circulantes en Colombia. Materiales y métodos. Se seleccionaron 96 aislamientos de pacientes con leishmaniasis cutánea o mucocutánea y se tipificaron mediante MLEE y PCR-RFLP con los blancos moleculares miniexon y hsp70 usados en serie. Las enzimas de restricción aplicadas fueron la HaeIII y la BccI, respectivamente. Se calculó el coeficiente kappa y un intervalo de confianza (IC) de 95 %. Resultados. Se determinó que la concordancia fue "muy buena" al obtener un coeficiente de 0,98 (IC95%: 0,98-1,00). Las especies identificadas fueron: Leishmania Viannia braziliensis, L. (V.) panamensis, L. (V.) guyanensis y L. (L,) amazonensis. De los 96 aislamientos, 80 se enviaron a secuenciación y se confirmaron los resultados obtenidos mediante PCR-RFLP. Conclusión. Dada la concordancia obtenida con la PCR-RFLP amplificando los genes miniexon y hsp70, se propone esta prueba como alternativa para la tipificación de especies de Leishmania circulantes en Colombia.

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress

Abstract Background: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. Methods: We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5 and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. Results: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. Conclusions: Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is noninduced by heat stress in S. neumayeri.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Hsp70 Knockdown by siRNA Decreased Collagen Production in Keloid Fibroblasts

PURPOSE: There are currently no consistently effective treatments for the excessive collagen produced by keloid fibroblasts. Previously, we reported that heat shock protein 70 (Hsp70) is up-regulated in keloid fibroblasts and keloid tissue. We, therefore, investigated whether Hsp70 is related to excessive collagen production in keloid fibroblasts. MATERIALS AND METHODS: We inhibited Hsp70 in keloid fibroblasts by RNA interference and examined the resulting collagen expression. Thus, we selected small interfering RNAs (siRNAs) specific for human Hsp70, transfected them into keloid fibroblasts, and evaluated the resulting phenotypes and protein production using real-time polymerase chain reaction (PCR), Western blot, and a collagen assay. RESULTS: The siRNAs dramatically suppressed Hsp70 mRNA expression, resulting in a decrease in collagen production in the keloid fibroblasts compared with controls. The siRNAs did not influence the viability of the keloid fibroblasts. CONCLUSION: Hsp70 overexpression likely plays an important role in the excessive collagen production by keloid fibroblasts. RNA interference has therapeutic potential for the treatment of keloids.

Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.

Effect of dexamethasone on unfolded protein response genes (MTJ1, Grp78, Grp94, CHOP, HMOX-1) in HEp2 cell line.

The endoplasmic reticulum (ER) is related to the various signal routes that are activated in unfolded protein response (UPR). The Grp78, Grp94, CHOP, MTJ1 and HMOX1 genes expressions demonstrate UPR activity. In this study, we investigated the UPR gene expressions in larynx epidermoid carcinoma (HEp2) to which dexamethasone (dex) was applied. HEp2 cells were administered for 48 h with different combinations using 0.1 μM and 1 μM dex, 1 mM phenyl butyric acid (PBA) and 100 ng/ml lipopolysaccharide (LPS). The Grp78, Grp94, CHOP, MTJ1 and HMOX1 genes expression was determined using quantitative RT-PCR. The Grp78, MTJ1 and HMOX1 gene expression increased with the administration of 1 µM dex. CHOP expression, on the other hand, decreased with 0.1 µM dex. When dex was combined with LPS, nearly all gene expressions decreased. The increase in Grp78, Grp94, HMOX1 and MTJ1 gene expression was greater in groups in which dex was administered in combination with PBA than in groups in which dex was administered alone. Dex in low dose (0.1 μM) caused a decrease in CHOP expression in HEp2 cells and an increase in Grp78 expression, in particular. The changes in UPR genes expressions may lead to the extended survival of the cells.

Antiaging, antistress and ROS scavenging activity of crude extract of Ocimum sanctum (L.) in Caenorhabditis elegans (Maupas, 1900).

Since aging is the most important risk factor for variety of diseases, the discovery of a wide range of chemical modulators of aging in model organisms encourages new strategies for targeting age associated diseases. Simple genetic manipulation leads to long-lived and healthy animals, so any compound which could have similar effect would prove a boon to mankind. In the present study, effect of different pharmacological doses (1.0, 0.1, 0.01 and 0.001 mg/mL) of O. sanctum crude extract were used to determine their impact on life span, thermotolerance and ROS scavenging activities in C. elegans. The results revealed that 1 mg/mL of O. sanctum extract significantly extended the life span of C. elegans. The extract also proved to be a strong free radical scavenger and increased resistance against thermal stress. It is also suggested that the protective and life span extending action of the crude extract is not only due to its antioxidant capacity but may also be mediated by modulation of some signaling pathways. Thus, in addition to all the known medicinal property of Ocimum, it is capable of increasing stress tolerance and life span in C. elegans.

Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Polymorphic analysis of MHClinked Heat Shock Protein 70 genes: Their susceptibility and prognostic implication in Kangri cancer cases of Kashmiri population.

Kangri cancer is a unique thermally-induced squamous cell carcinoma (SCC) of skin that develops due to persistent use of Kangri (a brazier), used by Kashmiri people, to combat the chilling cold during winter months. We designed a large scale case-control study to characterize the frequency of two polymorphisms within the MHC class III-linked HSP70genes, Hsp70-2 and Hsp70-hom, in order to find any association of these genotypic variants for predisposition to and clinical outcome of Kangri cancer patients from Kashmir valley in North India. Polymerase Chain Reaction and restriction enzymes were utilized to characterize the frequency of two polymorphisms with in Hsp70-2 and Hsp70-hom genes in 118 Kangri carcinoma cases and 95 healthy controls from the same population of Kashmir. Association of high frequency allelic variants of Hsp70genes with various clinicopathological features of prognostic significance was assessed by Chi-square test using SPSS software. In this study, allelic frequency of Hsp70-2 A/G heterozygote (0.87) (P = 0.012) was found to be significantly high in Kangri cancer cases compared to control (0.736) with a Relative Risk of 2.45 fold. Conversely, the allelic frequency of Hsp70-2 A/A allele in homozygous condition was significantly low in Kangri cancer cases and worked out to be 0.084 (Vs 0.252 in control) with P is equal to 0.001, implicating it as a protective allele against Kangri cancer in subjects with this genotype. Similarly, significantly high frequency of 0.50 (Vs 0.29 in control) of Hsp70-homC/C allele was found in homozygous condition in Kangri cancer cases suggestive of a positive relative risk associated with this genotype (RR is equal to 2.47) (P is equal to 0.002). The overall allele frequency data analysis of Hsp70-2 and Hsp70-hom genes was significant (χ2 is equal to 12.38, P is equal to 0.002; and χ2 is equal to 12.21, P is equal to 0.002). The study also reveals considerable association of high frequency alleles of HSP70 genes, especially of Hsp70-2 A/G or G/G in Kangri tumors with clinico-pathological features of poor prognosis. These results indicate that the relative risk of Kangri cancer associated with Hsp70-2 and Hsp70- hom gene polymorphisms is confined to Hsp70-2 A/G or G/G and Hsp70homC/C haplotype in our population. The study, therefore, suggests Hsp70-2 A/G or G/G and Hsp70homC/C genotypes as potential susceptibility markers and independent prognostic indicators in Kangri carcinoma patients in Kashmiri population.

Heat shock protein 70 alters the endosome-lysosomal localization of huntingtin

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. N- terminal fragments of huntingtin with polyglutamine produce aggregates in the endosome-lysosomal system, where proteolytic fragments of huntingtin is generated. Heat shock protein 70 (HSP70) prevents the formation of protein aggregates, but the effect of HSP70 on the huntingtin in the endosome-lysosomal system is unknown. This study was to determine whether HSP70 alters the distribution of huntingtin in endosome-lysosomal system. HSP70 expressing stable cells (NIH/3T3 or cerebral hybrid cell line A1) were generated, and mutant [(CAG)100] huntingtin was transiently overexpressed. Analysis of subcellular distribution by immnuocytochemistry or proteolysis cleavage by Western blotting was performed. 18 CAG repeat wild type [WT; (CAG)18] huntingtin was used as a control. Cells with huntingtin showed patterns of endosome- lysosomal accumulation, from a 'dispersed vacuole (DV)' type into a coalescent 'perinuclear vacuole (PV)' type over time. In WT huntingtin, HSP70 increased the cells with the PV types that enhanced the proteolytic cleavage of huntingtin. However, HSP70 reduced cells of the DV and PV types expressing mutant huntingtin, that result in less proteolysis than that of control. In addition, intranuclear inclusions were formed only in mutant cells, which was not affected by HSP70. These results suggest that HSP70 alters the distribution of huntingtin in the endosome-lysosomal system, and that this contributes to huntingtin proteolysis.

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

The inducible 70 kDa heat shock proteins (Hsp70) in mice are encoded by two almost identical genes, hsp70.1 and hsp70.3. Studies have found that only hsp70.1 is induced by hypertonic stress while both hsp70.1 and hsp70.3 genes are expressed in response to heat shock stress. It is unclear if the human counterparts, hsp70-2 and hsp70-1, are differentially regulated by heat shock and osmotic stress. This study found that only hsp70-2 was induced by hypertonic stress in human embryonic kidney epithelial cells and fibroblasts, while heat shock stress induced both hsp70-1 and hsp70-2. The human hsp70-2 promoter region contains three TonE (tonicity-responsive enhancer) sites, which were reported to play an important role in the response to hypertonicity. When the reporter plasmids containing different parts of the 5' flanking region of hsp70-2 were transfected into human embryonic kidney epithelial cells or fibroblasts, one TonE site at -135 was found to play a key role in the response to hypertonicity. The inactivation of the TonE site using site-directed mutagenesis led to the complete loss of induction by hypertonicity, which demonstrates the essential role of the TonE site. This suggests that the TonE site and the TonEBP (TonE binding protein) are the major regulators for the cellular response against high osmolarity in human kidney tissue.

Expression of heat stress protein 70 mRNA in patients with chronic obstructive pulmonary disease and its significance / 华中科技大学学报（医学）（英德文版）

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70 (Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0.24 +/- 0.11 and 0. 42 +/- 0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P < 0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9 +/- 9.9 vs 44.8 +/- 15.3, P < 0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0.85, P < 0.01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

Ecologically relevant stress resistance: from microarrays and quantitative trait loci to candidate genes - a research plan and preliminary results using Drosophila as a model organism and climatic and genetic stress as model stresses.

We aim at studying adaptation to genetic and environmental stress and its evolutionary implications at different levels of biological organization. Stress influences cellular processes, individual physiology, genetic variation at the population level, and the process of natural selection. To investigate these highly connected levels of stress effects, it is advisable - if not critical - to integrate approaches from ecology, evolution, physiology, molecular biology and genetics. To investigate the mechanisms of stress resistance, how resistance evolves, and what factors contribute to and constrain its evolution, we use the well-defined model systems of Drosophila species, representing both cosmopolitan species such as D. melanogaster with a known genome map, and more specialized and ecologically well described species such as the cactophilic D. buzzatii. Various climate-related stresses are used as model stresses including desiccation, starvation, cold and heat. Genetic stress or genetic load is modelled by studying the consequences of inbreeding, the accumulation of (slightly) deleterious mutations, hybridization or the loss of genetic variability. We present here a research plan and preliminary results combining various approaches: molecular techniques such as microarrays, quantitative trait loci (QTL) analyses, quantitative PCR, ELISA or Western blotting are combined with population studies of resistance to climatic and genetic stress in natural populations collected across climatic gradients as well as in selection lines maintained in the laboratory.

Protective Effect of Heat Shock Protein 70 Against Oxidative Stresses in Human Corneal Fibroblasts

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.

Effect of Thermal Preconditioning Before Excimer Laser Photoablation

The purposes of this study were to assess the expression patterns of heat shock proteins (Hsps), after eyeball heating or cooling, and to elucidate their relationships with corneal wound healing and intraocular complications after excimer laser treatment. Experimental mice were grouped into three according to local pretreatment type: heating, cooling, and control groups. The preconditioning was to apply saline eyedrops onto the cornea prior to photoablation. Following photoablation, we evaluated corneal wound healing, corneal opacity and lens opacity. Hsp expression patterns were elucidated with Western blot and immunohistochemical staining. The heating and cooling groups recovered more rapidly, and showed less corneal and lens opacity than the control group. In the heating and cooling groups, there were more expressions of Hsps in the cornea and lens than in the control group. These results were confirmed in the Hsp 70.1 knockout mouse model. Our study showed that Hsps were induced by the heating or cooling preconditioning, and appeared to be a major factor in protecting the cornea against serious thermal damage. Induced Hsps also seemed to play an important role in rapid wound healing, and decreased corneal and lens opacity after excimer laser ablation.