Hsp70 Research Articles

Novel SNP identification in exon 3 of HSP90AA1 gene and their association with heat tolerance traits in Karan Fries (Bos taurus × Bos indicus) cows under tropical climatic condition.

Heat shock proteins (HSPs) act as molecular chaperones those are preferentially transcribed in respose to heat stress and the polymorphism in HSP genes associated with heat tolerance traits in cows. HSP90AA1 gene has been mapped on Bos taurus autosome 21 (BTA-21) and spans nearly 5368 bp comprising of 11 exons out of which the first exon does not translate. The present study was done on Karan Fries (5/8 HF × 3/8 Tharparkar) cows reared in tropical climate with the objectives of identifying single-nucleotide polymorphisms (SNPs) in targeted regions (exon 3) of HSP90AA1 gene and analyzing their association with heat tolerance traits in Karan Fries cows. Respiration rate (RR) and rectal temperature (RT) were recorded once daily for four consecutive days during probable extreme hours in different seasons or temperature humidity index (THI), viz., winter, spring, and summer. For detecting single-nucleotide polymorphisms, sequence data were analyzed using BioEdit software (version 7.2). Comparative sequence analysis of HSP90AA1 gene showed point mutation, viz., g.1209A>G (exon 3) as compared to Bos taurus (NCBI Ref Seq: AC_000178.1). Association analysis indicated that THI was influenced (P < 0.01) by RR, RT, and HTC. Similarly, SNPs at locus g.1209A>G were categorized into three genotypes, i.e., AA, AG, and GG, and the least squares means (LSMEANS) of RR, RT, and HTC for GG (homozygous) genotype were significantly lower (P < 0.01) than AA (homozygous) and AG (heterozygous) genotypes. These findings may partly suggest that cows with GG genotypes were favored for heat tolerance trait, which can be used as an aid to selection for thermo-tolerance Karan Fries cows for better adaptation in subtropical and tropical hot climate.

Tolerogenic responses of CD206+, CD83+, FOXP3+, and CTLA-4 to sericin/polyvinyl alcohol/glycerin scaffolds relevant to IL-33 and HSP60 activity.

Silk sericin-releasing (sericin/polyvinyl alcohol (PVA)/glycerin) scaffolds have been designed for wound dressing applications using different fabrication techniques that influence scaffold antigenicity. The immunological tolerance of scaffolds depends on the balance of immunogenic and tolerogenic responses modulated by dendritic cells (DCs). An in vivo skin implantation model was used to compare the tolerogenic effect of sericin/PVA/glycerin scaffolds prepared by freeze-drying versus salt-leaching techniques, using an Allevyn® scaffold as a control. Immunohistochemical and histopathological studies were performed to evaluate tolerogenic DCs (CD206+), immunogenic DCs (CD83+), regulatory T-cells (FOXP3+ and CTLA-4), a proinflammatory cytokine (interleukin 33: IL-33), a stress marker (heat shock protein 60; HSP60), histopathological changes and related inflammatory cells. It was found that both sericin/PVA/glycerin scaffolds were tolerogenic and induced early activated Treg functions, while the Allevyn® scaffold was immunogenic. However, the tolerance of the freeze-dried sericin/PVA/glycerin scaffolds was not as consistent as the salt-leached sericin/PVA/glycerin scaffolds, indicated by the low level of CTLA-4 expression. This was probably due to molecular cross-linking and the morphological and mechanical properties of the freeze-drying technique, which would enhance the immune response. Severe inflammatory responses (including mast cell degranulation and foreign body giant cell accumulation) and histopathological changes (including fat infiltration and fibrosis formation) were mainly found with the Allevyn® scaffold, presumably from its architecture and chemical composition, especially polyurethane. The up-regulation of IL-33 and HSP60 with the Allevyn® scaffold was correlated with the inflammatory and pathological levels. Our findings suggested that salt-leached sericin/PVA/glycerin scaffolds were tolerogenic, induced a low inflammatory response and were appropriate for wound dressing applications.

In silico analysis of regulatory and structural motifs of the ovine HSP90AA1 gene.

Gene promoters are essential regions of DNA where the transcriptional molecular machinery to produce RNA molecules is recruited. In this process, DNA epigenetic modifications can acquire a fundamental role in the regulation of gene expression. Recently, in a previous work of our group, functional features and DNA methylation involved in the ovine HSP90AA1 gene expression regulation have been observed. In this work, we report a combination of methylation analysis by bisulfite sequencing in several tissues and at different developmental stages together with in silico bioinformatic analysis of putative regulating factors in order to identify regulative mechanisms both at the promoter and gene body. Our results show a "hybrid structure" (TATA box + CpG island) of the ovine HSP90AA1 gene promoter both in somatic and non-differentiated germ tissues, revealing the ability of the HSP90AA1 gene to be regulated both in an inducible and constitutive fashion. In addition, in silico analysis showed that several putative alternative spliced regulatory motifs, exonic splicing enhancers (ESEs), and G-quadruplex secondary structures were somehow related to the DNA methylation pattern found. The results obtained here could help explain the differences in cell-type transcripts, tissue expression rate, and transcription silencing mechanisms found in this gene.

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (−74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT2 Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.

HSP60 as a novel autoantigen in obesity

HSP60 as a novel autoantigen in obesity

Escherichia coli under Ionic Silver Stress: An Integrative Approach to Explore Transcriptional, Physiological and Biochemical Responses.

For a better understanding of the systemic effect of sub-lethal micromolar concentrations of ionic silver on Escherichia coli, we performed a multi-level characterization of cells under Ag+-mediated stress using an integrative biology approach combining physiological, biochemical and transcriptomic data. Physiological parameters, namely bacterial growth and survival after Ag+ exposure, were first quantified and related to the accumulation of intracellular silver, probed for the first time by nano secondary ion mass spectroscopy at sub-micrometer lateral resolution. Modifications in E. coli biochemical composition were evaluated under Ag+-mediated stress by in situ synchrotron Fourier-transform infrared microspectroscopy and a comprehensive transcriptome response was also determined. Using multivariate statistics, correlations between the physiological parameters, the extracellular concentration of AgNO3 and the intracellular silver content, gene expression profiles and micro-spectroscopic data were investigated. We identified Ag+-dependent regulation of gene expression required for growth (e.g. transporter genes, transcriptional regulators, ribosomal proteins), for ionic silver transport and detoxification (e.g. copA, cueO, mgtA, nhaR) and for coping with various types of stress (dnaK, pspA, metA,R, oxidoreductase genes). The silver-induced shortening of the acyl chain of fatty acids, mostly encountered in cell membrane, was highlighted by microspectroscopy and correlated with the down-regulated expression of genes involved in fatty acid transport (fadL) and synthesis/modification of lipid A (lpxA and arnA). The increase in the disordered secondary structure of proteins in the presence of Ag+ was assessed through the conformational shift shown for amides I and II, and further correlated with the up-regulated expression of peptidase (hfq) and chaperone (dnaJ), and regulation of transpeptidase expression (ycfS and ycbB). Interestingly, as these transpeptidases act on the structural integrity of the cell wall, regulation of their expression may explain the morphological damage reported under Ag+-mediated stress. This result clearly demonstrates that the cell membrane is a key target of ionic silver.

Single nucleotide polymorphisms in Heat Shock Protein (HSP) 90AA1 gene and their association with heat tolerance traits in Sahiwal cows

Heat Shock Proteins (HSPs) are group of proteins which are synthesized during heat stress. HSP genes have been reported to be associated with heat tolerance and production performance in cattle. HSP90AA1 gene has been mapped on Bos taurus autosome 21 (BTA 21) and spans nearly 5368 bp comprising 11 exons out of which first exon does not translate. The present study was carried out in Sahiwal cows (n=100) with the objectives to identify SNPs in targeted regions (exon 3, 7, 8 &amp; 11) of HSP90AA1 gene and to analyze their association with heat tolerance traits in Sahiwal cows. Respiration rate (RR) and rectal temperature (RT) was recorded once during probable extreme hours in winter, spring and summer season. Further, heat tolerance coefficient (HTC) also calculated to see the adaptability of the animals during the period of heat stress. On the basis of comparative sequence analysis, total five SNPs were revealed at position of A1209G, A3292C, T4935C, T5218C and A5224C in the targeted region of HSP90AA1 gene. Out of these, only two SNPs at A1209G and A3292C loci were found significantly associated with heat tolerance traits in Sahiwal cows. Whereas, THI has a highly significant associated with RR, RT and HTC in all the seasons. At A1209G locus in Sahiwal cows for traits RR, genotype AA (18.40±0.46a), AG (19.60±0.85b) and GG (21.18± 0.64ab) and trait HTC AA (1.78±0.04a), AG (1.85±0.03b) and GG (1.91± 0.02c) differ significantly (p&lt;0.01) while trait RT, AG (38.32±0.10b) and GG (38.27±0.08ab) didn’t differ significantly. For a locus A3292C for traits RR, genotype AA (21.55±1.01a), AC (19.66±1.59b) and CC (18.40±1.03c) differ significantly (p&lt;0.05), while traits RT, genotype AA (38.41±0.12), AC (38.28±0.20) and CC (37.96±0.15) didn’t differ significantly. For trait HTC, AA (1.93±0.04a), AC (1.85±0.06b) and CC (1.79±0.05c) found significantly (p&lt;0.05) differ with each other. Our study indicated that Sahiwal cows of AA and AC genotype had better thermo-tolerance capacity, which had been useful for genetic improvement of Sahiwal cattle for heat tolerance traits.

The expressions of Caspase-9 and heat-shock protein-90 after focal cerebral ischemia-reperfusion injury in rats

Objective To investigate the expression of Caspase-9 and heat-shock protein-90 (HSP 90) in rats after focal cerebral ischemia-reperfusion injury. Methods The male SD rats (200-250 g) were divided into three groups by the random number table: normal group, sham group and cerebral ischemia-reperfusion (CIR) group. Each group was sorted into four subgroups including group 6 h, group 24 h, group 48 h and group 72 h according to the reperfusion time. Suture-occluded method was adopted to prepare focal cerebral ischemia-reperfusion(CIR) injury in rat model. Enzyme-linked immunosorbent assay (ELISA) method was used to detect the variations of Caspase-9 and HSP-90 expression in rats. Results The changes in Caspase-9 and HSP 90 expression in the brain cells were observed by ELISA method. The expression of Caspase-9 and HSP-90 was weakly expressed in sham group, and was at peak in CIR group within 24 h-48 h, then began to decline at 72 h after the reperfusion time. The differences in the expression of caspase-9 and HSP-70 between sham group and normal group were not statistically significant. Conclusions Apoptotic cells gradually increase along with reperfusion time and reach the peak at 48 h after cerebral ischemia-reperfusion. In ischemia half dark stripe, the expression of Caspase-9 and HSP 90 is increased in neuronal cells after cerebral ischemia-reperfusion, and the positive cells number is at peak at 48 h after cerebral ischemia-reperfusion. Apoptosis of neuronal cells after cerebral ischemia and reperfusion is a dynamic evolutionary process. The expression of Caspase-9 and HSP 90 in nerve cells plays an important role in regulating cell apoptosis. Key words: Reperfusion injury; Caspase-9; Heat-shock proteins

Inactivating Heat Shock Factor 1 (HSF1) in Acute Myeloid Leukemia By Pharmacological Inhibition of eIF4a: A Promising Therapeutic Approach

Heat shock factor 1 (HSF1) is best known as a key sensor of proteotoxic stress, but accumulating evidence also supports a major role for this transcriptional regulator in cancer biology. In a variety of human solid tumor cells, downregulation of HSF1 inhibits growth, induces cell death and limits metastatic potential. In breast cancers, nuclear accumulation of HSF1 and a tumor-specific gene expression signature reflecting HSF1 activation were found to be strongly associated with poor outcome (Mendillo et al, Cell 2012). In addition, we have recently reported, as a counter-intuitive reversal of the central dogma, that inhibition of protein translation represses the constitutive activation of HSF1 in cancers, and that HSF1 inhibition induced by the potent eIF4a inhibitor rohinitib (RHT) exerts profound, far-ranging anti-tumor effects (Santagata et al, Science 2013). Review of public databases supports targeting of HSF1 and eIF4a in AML: mRNA levels of HSPA8, one of the primary HSF1 targets, are correlated with poor prognosis in AML (Prognoscan, data from Metzeler et al, Blood 2008) and eIF4a mRNA levels were highest in AML among 12 cancer types (Oncomine, data from Ramaswamy et al, PNAS 2001). Here, we demonstrate that inactivation of HSF1 in acute myeloid leukemias (AMLs) by RHT exerts pronounced apoptogeniceffects with preferential activity against FLT3-ITD mutant cells in cell culture and in mice.First, we confirmed our previous finding of inactivation of HSF1 by RHT in AML. In OCI-AML3, MOLM-13 and MV4;11 cells, mRNA levels of HSPA8 were reduced by 70% after RHT treatment compared to untreated controls. OCI-AML3 cells were then infected with lentivirus encoding a reporter GFP-luciferase fusion protein the expression of which is driven by promoter elements from either the HSPA1A or HSPA6 genes; an approximately 50% reduction of reporter induction by heat shock was observed after RHT treatment compared to untreated controls. Next, treatment of 7 human AML cell lines in culture showed that RHT induces marked anti-leukemia effects at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity as determined by flow cytometry at 72hr). The most pronounced cytotoxic effects were observed in FLT3-ITD+ cell lines (LD50s < 10 nM in MOLM13 and MV4;11 cells). Using two sets of isogenic cell lines (Ba/F3 and OCI-AML3 cells with FLT3-ITD or wild-type (wt) FLT3), we confirmed that RHT more potently kills FLT3-ITD cells (LD50s; 65.3 vs 20.1 nM in Ba/F3 cells). Furthermore, the combination of FLT3 inhibitor sorafenibwith RHT showed synergistic effects in cell culture (Combination Index: ED50 0.85, ED75 0.86, ED90 0.89). Immunoblot analysis showed higher phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 cells than FLT3-wt cells, suggesting greater dependence of FLT3-ITD cells on HSF1 activation for survival. We also tested primary samples from 17 AML patients and bone marrow (BM) samples from 8 healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (Figure 1A). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem cells (CD45+CD34+CD38-). Moreover, the activity of RHT against the leukemic population was significantly higher in FLT3-ITD than in FLT3-wt cells (Figure 1B). We also evaluated the activity of RHT in a FLT3 mutant AML xenograft model using GFP-luciferase labeled MOLM-13 cells. Significantly decreased luciferase activity was detected by bioluminescence imaging and a dose-dependent reduction in GFP+ leukemic cells was seen in peripheral blood and BM by day 16 (Figure 2). Survival of the treatment groups was significantly prolonged (median; 18 vs 22.5 vs 24 days respectively, p < 0.0001).In conclusion, HSF1 function provides an attractive therapeutic target in AML. The eIF4a inhibitor RHT down-regulates HSF1 transcriptional function and exerts robust anti-leukemia activity in cell culture and in mice. Although the relative contributions of HSF1 inactivation and translation inhibition to the net anti-leukemic activity of RHT remain to be defined, promising features of this approach include its activity against AML stem cells, while sparing normal stem cells and its particularly potent cytotoxicity for poor-prognosis FLT3-ITD AMLs. Taken together, these preclinical findings strongly support further development of eIF4a inhibitors in the treatment of AML. [Display omitted] [Display omitted] DisclosuresIshizawa:Karyopharm: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Low-Dose Ionizing Radiation Modulates Expression of Gpam in Immortalized EBV Infected B Cells through miRNA Network

Low-dose ionizing radiation (LDIR, ≤ 0.1 Gy) which is typically associated with therapeutic and diagnostic radiological modalities, is known to induce remodeling of the stromal microenvironment. Whereas the biologic responses to LDIR are commonly described as a stress response, the carcinogenic potential of this environmental stressor remains unknown. Recently, ionizing radiation (IR)-induced alterations of miRNA expression that play a fundamental role in cell signaling events, have been demonstrated in various cell types (Marsit, Cancer Res. 2006).To assess a potential determinant influencing of LDIR induced miRNA alterations in pre-malignant cells in a microenvironment, we utilized immortalized pre-malignant Epstein-Barr virus infected-B (EBV-B) cells which are continuously proliferating in circulation or in lymph nodes. The LDIR system (0.1 Gy, 4MV X ray from a LINAC) was utilized in the in vitro co-culture of EBV-B and mesenchymal stromal cells (MSC) to mimic the lymph node stromal microenvironment. We confirmed that MSC protected co-cultured EBV-B cells from spontaneous apoptosis and caused accumulation of EBV-B cells in the G0/G1 phase (EBV-B; monoculture vs coculture with MSC; Sub G1% 42.0±2.4 vs 34.1±0.9 p< 0.01, G0/G1 % 42.6±2.1 vs 47.6±2.0, p<0.05).To identify LDIR-induced modulation of specific miRNAs in EBV-B cells, a high-confidence list of 44 known LDIR-associated miRNAs from three separate screening studies of lymphoblastic cells was assembled (Cha et al., Oncol Rep.2009, Chaudhry et al., J Biomed Sci. 2010, Lhakhang et al., Comp Funct Genomics. 2012). From these miRNA, we focused on four miRNAs let-7a, miR-16, miR-19b and miR-21 that have been listed in more than two of the screens. Our studies showed that LDIR upregulated let-7a and miR-16 levels in irradiated mono-cultured EBV-B, but on the contrary downregulated all tested miRNAs in MSC co-cultured EBV-B cells (at 24-hour).We next investigated alterations of EBV-B gene expression by LDIR using the DNA microarray (Affymetrix). In mono-cultured EBV-B cells, cDNA microarray analysis detected upregulation of TGFB1 mRNA, and downregulation of the genes encoding a lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase (GPAM), a growth factor amphiregulin (AREG), cell survival-/growth-related factors Rho-GTPase effector gene folmin-1 (FMN1), chemotaxis-inducing chemokine gene IL-8 and B-cell lymphoma 2 gene BCL2, all of which were confirmed by qRT-PCR. Downregulation of the GPAM protein was further shown on the protein level by western blot, and the ontology analysis demonstrated that LDIR caused the TGFbeta-dependent induction of the epithelial-mesenchymal transition (EMT) pathway in mono-cultured EBV-B cells. In contrast to the mono-culture condition, the GPAM mRNA and protein expression were upregulated in MSC co-cultured EBV-B cells.To determine the potential targets of LDIR-altered miRNAs, we next identified 79 genes that are commonly targeted by let-7a, miR-16, miR-19b, or miR-21 from a microrna.org database, and found that the expression changes of GPAM mRNA and protein were strikingly matched with miRNA profiling. Partial concordant changes of DNAJ(HSP40)A2 coding heat shock protein (HSP) 40 homolog, a cochaperone of HSP70s, CPEB3 coding RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CEBP3) and its transcriptional target GLUR2 (glutamate receptor subunit), a receptor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were also observed in the microarray results.Since GPAM is a lipid-related metabolism gene which is known to be regulated by miR-27b and to negatively impact survival of breast cancer cells we next investigated the expression changes of miR-27b after LDIR in EBV-B cells. As expected, LDIR upregulated miR-27b in mono-cultured EBV-B cells, and downregulated in co-cultured EBV-B cells with MSC. Intriguingly, no significant changes of GPAM mRNA as well as tested miRNA expression was observed by 1Gy irradiation.In summary, we demonstrated that LDIR directly modulates EBV-B cells gene expression. Importantly, LDIR additionally impacts EBV-B cells indirectly through miRNA modulation by the neighboring stromal cells. DisclosuresKonopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

The Hsp90 inhibitor FW-04-806 suppresses Bcr/Abl-mediated growth of leukemia cells by inhibiting proliferation and inducing apoptosis

To investigate the antitumor efficacy and mechanism of HSP90 inhibitor FW-04-806 against Bcr/Abl(+) leukemia K562 and HL60 cells and their mechanisms of action. MTT assay was used to assess the proliferation-inhibiting effect of FW-04-806. Cell cycle was analyzed with propidium iodide by flow cytometry. Cell apoptosis was determined using the FITC mV apoptosis detection kit. Western blot was applied to reveal the protein expression of related proliferative and apoptotic signaling pathways. The changes of mitochondrial membrane potential were detected by flow cytometry. Protein-protein interactions was shown by co-immunoprecipitation. The level of mRNA was assessed by real-time RT-PCR. FW-04-806 obviously inhibited cell proliferation in the HL60, K562 and HL60/Bcr-Abl cell lines, with an IC50 of (30.89 ± 0.12) µmol/L, (9.76 ± 0.19) µmol/L and (8.03 ± 0.26) µmol/L, respectively (P<0.001). Compared with the vehicle group, the two increasing doses of FW-04-806 showed inhibition of tumor growth at a rate of (17.40 ± 0.34)% and (34.33 ± 5.00)%, respectively, in the K562 cell line groups (P=0.003), and (18.90 ± 1.45)% and (35.60 ± 3.55)% (P=0.001) in the HL60/Bcr-Abl cell line groups. FW-04-806 dissociated Hsp90/Cdc37 chaperon/co-chaperon complex, followed by degradation of the Hsp90 proteins through proteasome pathway without affecting mRNA expression. FW-04-806 induced apoptosis and led to G2/M arrest. Our findings indicate that FW-04-806 displays potential antitumor effect by suppressing the proliferation and apoptosis in Bcr/Abl(+) leukemia cells in vivo.

Immunoexpression patterns for Hypoxia-inducible Factor-1α and von Hippel-Lindau protein, in relation to Hsp90, of human brain tumors.

The pathogenesis of many tumors, including brain tumors, has been associated with hypoxia, which induces the transcriptional activity of hypoxia-inducible Factor-1α (HIF-1α). HIF-1α is normally degradated by the von Hippel-Lindau protein (pVHL) but, in hypoxia, pVHL/HIF-1α interaction is inhibited resulting in the nuclear accumulation of HIF-1α. Hsp90 (Heat shock protein 90), as a chaperone protein, plays a critical role for both stabilization of HIF-1α and degradation of pVHL. The aim of this study was to estimate immunohistochemically the expression levels of HIF-1α and pVHL, in relation to Hsp90, in different types of human brain tumors (42 gliomas, 9 medulloblastomas, and 38 meningiomas) using specific antibodies. The tumors were further divided into two groups according to the age of patients (≥19 years old or ⟨19 years old). Nuclear, for HIF-1α, and cytoplasmic, for pVHL and Hsp90, localization was detected in a high percentage of tumor cells in the majority of tumors. In astrocytomas, a significant, grade-dependent relationship for HIF-1α immunoexpression was observed (p⟨0.05). Furthermore, there was a significant correlation between pVHL and Hsp90 immunoexpression (p⟨0.01). The group of ≥19 years old patients with glioblastomas (WHO grade IV) demonstrated significantly increased immunoexpression for HIF-1α compared to pVHL (p⟨0.0001) and Hsp90 expression (p⟨0.01). In medulloblastomas, a significant correlation of HIF-1α with Hsp90 immunoexpression (p⟨0.05) was found. In meningiomas, no significant correlation for the expression of the three proteins was detected (p≥0.05). These results indicate that HIF-1α/pVHL/Hsp90 interactions may be implicated in biology of different types of brain tumors through different signaling mechanisms.

Relationship between human heat shock protein 60 IgG from porphyromonous gingivalis and coronary heart disease in the elderly

Objective To investigate the relationships of human heat shock protein 60 (HSP 60), heat shock protein 60 IgG (HSP60 IgG), heat shock protein 60 IgG from porphyromonous gingivalis (P.g HSP60 IgG) with coronary heart disease and periodontitis, in order to provide the theoretical basis on the new risk factors for coronary heart disease. Methods A total of 128 veteran cadres who took health physical examination in the Second Hospital of Tianjin Medical University from September to October in 2012 were recruited. They were divided into 4 groups: the healthy control group (n=30), coronary heart disease group (CHD group, n=27), the periodontitis group (n=36), CHD combined with periodontitis group (n=35). The concentrations of HSP60, HSP60 IgG, P. g HSP60 IgG were determined by enzyme-linked immunosorbent assay (ELISA), and the general biochemical markers such as liver and kidney function, blood lipids were detected at the same time. Results The concentrations of human HSP60, HSP60 IgG, P. gHSP60 IgG were higher in CHD, periodontitis and CHD combined with periodontitis groups than in the healthy control group (all P<0.05). The above indicators in coronary heart disease combined with periodontitis group were also increased compared with those in CHD and periodontitis groups (all P<0.05). The patients with periodontitis had higher P. g HSP60 IgG concentration than patients with CHD (P<0.05). The partial correlation analysis showed that HSP60 and P. g Hsp60 IgG levels were positively correlated with human HSP60IgG level (r=0.705, 0.825, P<0.01). Multivariate logistic regression analysis showed that human HSP60, HSP60IgG and P. gHsp60IgG may be the risk factors for coronary heart disease (all P<0.05). Conclusions Human HSP60, HSP60 IgG and P. gHSP60 IgG may play a bridging role between coronary heart disease and periodontitis. The elderly patients with periodontitis may be more susceptible to coronary heart disease. Key words: Periodontitis; Coronary disease; Heat-shock proteins; Porphyromonas, endodontalis

Genome-wide Identification and Expression Pattern Analysis of the DnaJ Protein Family in Gossypium raimondii under Salt Stress Using a Bioinformatics Method

Genome-wide Identification and Expression Pattern Analysis of the DnaJ Protein Family in Gossypium raimondii under Salt Stress Using a Bioinformatics Method

Exercise preconditioning attenuates pressure overload-induced pathological cardiac hypertrophy: potential role of HSF1 and NF-κB p65 signaling

To observe the effect of exercise preconditioning (EP) on pressure overload-induced pathological cardiac hypertrophy and explore related mechanisms. Ten-week-old male Sprague-Dawley rats (n = 80) were randomly divided into four groups via random number table method: sham, TAC, EP + sham and EP + TAC. Two EP groups were subjected to 4 weeks of treadmill training, and followed by sham and TAC operations. Eight weeks after the surgery, mean arterial pressure (MAP), cardiac morphology, mRNA expressions of the B-type natriuretic peptide (BNP) and heat shock protein (HSP) 70 and protein expression of the BNP, heat shock transcription factor 1 (HSF1), HSP70, nuclear factor κB (NF-κB) p65, and interleukin-2 (IL-2) were examined. (1) Pathological cardiac hypertrophy index: eight weeks after TAC, MAP, heart size, HW/BW, cross-sectional area of the cardiomyocytes (CSA) and mRNA and protein expressions of BNP in the LV were all significantly higher in the TAC and EP + TAC groups than respective sham groups (all P < 0.05). HW/BW, CSA, and mRNA and protein expressions of BNP in the LV were significantly lower in EP + TAC group than in TAC group (all P < 0.05). (2) mRNA and protein expressions of HSF1 and HSP70 and nuclear HSF1 levels were significantly downregulated post TAC, however, EP treatment significantly increased the expression of HSF1 and nuclear HSF1 levels in TAC rats (all P < 0.05). (3) mRNA and protein expressions of NF-κB p65 and IL-2 were significantly increased in the TAC and EP + TAC groups compared with the respective sham groups (all P < 0.05), which were significantly downregulated in EP + TAC group compared to TAC group (all P < 0.05). EP could effectively reduce the cardiac hypertrophic responses induced by TAC possibly through upregulating the expressions of HSF1 and HSP70 and inhibiting the expression of NF-κB p65 and its nuclear translocation.

Quercetin and hydroxytyrosol attenuates xanthine/xanthine oxidase-induced toxicity in H9c2 cardiomyocytes by regulation of oxidative stress and stress-sensitive signaling pathways.

The increased activity of xanthine/xanthine oxidase (X/XO) has been suggested as a risk factor for heart disease and herbal polyphenols exhibits cardioprotection in vitro and in vivo. To understand the cardioprotective action mechanisms of polyphenol quercetin and hydroxytyrosol, the expression levels of stress-responsive proteins were studied in X/XO-induced toxicity model of H9c2 cardiomyocyocytes. Pretreatment with each polypenol (0.1-10 μg/ml; 24 h) enhanced viability (p < 0.01; MTT test) and inhibited reactive oxygen species (ROS) generation (p < 0.001; H2DCFDA assay) against 12 h exposure to a free radical generating system, X (0.5 mM) and XO (5 mU/ml). Western blotting experiments showed that X/XO increases the phosphorylation of downstream substrate of p38, MAPK-activated protein kinase 2 (MAPKAPK-2), p44/42-MAPK (Erk1/2) and cleaved caspase-3 (p < 0.001, vs. Control), however inhibits the levels of phosphorylated c-Jun and Hsp27 (p < 0.01, vs. Control). Pretreatment with quercetin or hydroxytyrosol attenuated the phosphorylation of MAPKAPK-2 and cleaved caspase-3 in X/XO-exposed cells (p < 0.01, vs. X/XO). Hydroxytyrosol enhanced the reduction of phosphorylation of a transcriptional target c-Jun and led to overphosphorylation in protective proteins, p44/42-MAPK and Hsp27 in X/XO-exposed cells (p < 0.01, vs. X/XO). Our data suggest that quercetin and hydroxytyrosol protects cardiomyocytes against X/XO-induced oxidative toxicity by diminishing intracellular ROS and the regulation of stress-sensitive protein kinase cascades and transcription factors.

Proteomics of differentially-expressed proteins between human primary and lymph node metastatic gastric adenocarcinoma

Objective To identify the differentially-expressed proteins of human gastric adenocarcinoma cell in primary or metastatic lymph node tissues by comparative proteomics technology, and to screen the specific metastatic-associated proteins so as to investigate the metastatic molecular mechanism of lymph node metastasis in gastric cancer. Methods 11differential proteins were acquired previously from primary and metastatic lymphnode tissues in gastric adenocarcinoma patients by 2D-DIGE. Some selected differential protein spots were identified by PMF based on MALDI-TOF-MS and database search. Immunohistochemical staining of HSP70 was used to evaluate the reliability of the proteomic analysis results. Results After analyzed on 11 differential proteins by in-gel trypsin digestion and MALDI-TOF-MS-based PMF analysis, a total of 5 differential proteins were identified by searching Mascot-database, HSP70's 8 isoform 2 variant, chaperonin, chaperonin, leucine aminopeptidase, predicted: hypothetical protein XP_515584. Among the differential proteins identified, the levels of HSP70, chaperonin, leucine aminopeptidase expression had a significant up-regulation in gastric primary cancer compared with metastatic lymph node. HSP70 expression rate increased with the metastasis of lymph node and the progress of gastric cancer, agreed with the proteomics results. Conclusions They are similar in differentially-expressed proteins in primary or metastatic lymph node tissues because of the uniformity in source and differention. There are few protein changes in cancer cells between them, taking part in the metastatic of gastric cancer. HSP70 takes part in the progress of gastric cancer and relates to the metastasis of lymph node and malignant degree. Key words: Stomach neoplasms; Adenocarcinoma; Lymphatic metastasis; Microdissection; Spectrometry, mass, matrix-assisted laser desorption-ionization; Proteomics

Chaperone DnaJ Influences the Formation of Biofilm by Escherichia coli

DnaJ chaperone, a member of the so called DnaK-DnaJ-GrpE chaperone machine plays an important role in cell physiology. The ability of Escherichia coli ΔdnaJ mutant to form biofilm was studied. It was shown that this mutant is impaired in biofilm development when exposed to 42 degrees C for 2 h. The impairment in biofilm development was observed when the heat shock was applied either at the onset of biofilm formation or 2 h later. The biofilm formed was thinner and its structure was changed as compared to wild-type strain. This defect could be complemented by the introduction of a wild-type gene on a low-copy plasmid.

The expression levels of serum HSP70 and P53 in gastric cancer and its correlation with helicobacter pylori infection

Objective To study the expression levels of serum HSP70 and p53 in gastric cancer and its correlation with helicobacter pylori infection. Methods 50 patients with gastric cancer were selected as the research object. Among 50 cases,30 cases were stageⅠ-Ⅱ, 20 cases were stage Ⅲ, 22 cases were with lymph node metasta-sis and 28 cases were without lymph node metastasis.40 healthy subjects were selected as the healthy control group. The serum levels of HSP70 and p53 were detected. Results In the gastric cancer group, the serum levels of HSP70 and p53 were markedly higher than those of the healthy control group, and the difference was statistically significant (t＝12.53,16.79,all P＜0.01). Moreover, with progressing of clinical stage of gastric cancer, the serum levels of HSP70 and p53 showed a trend of increasing(t＝4.68,5.29,all P＜0.01). The serum expression levels of HSP70 and p53 in gastric cancer with lymph node metastasis were significantly higher than those of patients without lymph node metastasis and the difference was still statistically significant(t＝3.82,4.39,all P＜0.01). The serum levels of HSP70 and p53 in the gastric cancer group with Hp positive were much higher, compared to those of cases with Hp negative and the difference was also statistically significant(t＝4.72,4.17,all P＜0.01). Conclusion In the gastric cancer patients, the serum expression level of HSP70 and p53 are increased. The expression of HSP70 and p53 are closely related to the progress and prognosis of gastric cancer and Hp infection. Key words: Stomach neoplasms; Helicobacter pylori; HSP70 Heat-shock proteins; Genes，p53

Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study.

Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.