Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd(2.).

The 70-kDa heat shock proteins (HSP70) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. The full-length HSP70 cDNA of Sepiella maindroni (designated as SmHSP70, GenBank accession no. KJ739788) was 2109 bp, including an ORF of 1950 bp encoding a polypeptide of 649 amino acids with predicted pI/MW 5.24/71.30 kDa, a 62 bp-5'-UTR and a 97 bp-3'-UTR. BLASTp analysis and phylogenetic relationship strongly suggested that the amino acid sequence was a member of HSP70 family. Multiple sequence alignment revealed that SmHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-binding site motif and 'EEVD' motif. Time-dependent mRNA expression of SmHSP70 in the liver was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmHSP70 played a significant role in mediating the environmental stress and immune response against pathogens.

Differential HSP70 expression in Mytilus coruscus under various stressors.

Heat shock proteins (HSPs) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. The full-length HSP70 cDNA of Mytilus coruscus (designated as McHSP70, GenBank accession no. KF322135) was obtained, which was 2319 bp, including an ORF of 1965 bp to encode a polypeptide of 655 amino acid with predicted pI/MW5.29/71.38 kDa, a 81 bp-5'-UTR and a 270 bp-3'-UTR. The BLAST analysis and phylogenetic relationship strongly suggested that this amino acid sequence was a member of HSP70 family and highly homologous with Mytilus galloprovincialis (95%). Multiple sequence alignment revealed that McHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-Binding site motif and 'EEVD' motif. The mRNA of McHSP70 in hemolymph was constitutively expressed in all treatments including Vibrio challenge, thermal stress, metals (copper and cadmium) and 180 CST fuel exposure based on SYBR Green quantitative RT-PCR analysis. The temporal expression of HSP70 mRNA in hemolymph was significantly affected by Vibrio alginolyticus and Vibrio harveyi challenge. The maximum level appeared at 24h post-injection with 5.44-fold in V. alginolyticus and dropped back to the original level at 72 h post-injection. In V. harveyi infection the transcripts of McHSP70 was significantly induced to the highest at 2h after post-injection with 13.52-fold, then decreased until 36 h appearing another expression peak with 10.29-fold, and returned gradually to physiological level at the end of this experiment. In heat shock experiment the maximum expression appeared at 12h with 15-fold higher than that of blank mussels. Time-dependent mRNA expression pattern of McHSP70 was found in exposure to copper, cadmium and 180 CST fuel, but the highest expression and time were different in various treatments (copper of 9.41-fold at day 15, cadmium of 10.82-fold at day 10, and 180 CST fuel of 5.94-fold at day 25), and then dropped progressively. All these results indicated that HSP70 in marine bivalve had a significant role in mediating the environmental stress and immune response.

[Effect of Shenkang Injection on Kidney Function in Hypertensive Renal Damage Rats].

OBJECTIVE: To observe the effect of Shenkang Injection on the blood pressure, metabolism, blood biochemistry and renal pathology in hypertension renal damge rats, then to provide theoretical basis for clinical trials. METHODS: 75 spontaneously hypertensive nephropathy rats were randomly divided into five groups with 15 rats in each group: model group (SHR group) rats were intragastrically treated with the vehicle (4 mL/kg normal saline per day) of Shenkang Injection per day; Benazepril group( positive control group, 8 mg/ kg Benazepril per day) ;Shenkang Injection low-dose group (6.7 mL/kg Shenkang Injection per day); middle-dose group (13.3 mL/kg Shenkang Injection per day); high-dose group (26.6 mL/kg Shenkang Injection per day); and WKY rats were normal control group (n = 15) (4 mL/kg normal saline per day). RESULTS: After 3 months intraperitoneal injection treatment, SHR rats blood pressure were in- hibited; the levels of microalbumin (m-ALB), total protein (U-TP), serum creatinine (Ser) and urea nitrogen (BUN) were decreased significantly in Shenkang Injection treated groups rats. Shenkang Injection significantly improved the levels of creatinine clearance rate (Ccr), serum albumin (ALB) and superoxide dismutase (SOD), decreased the content of methane dicarboxylic aldehyde (MDA), aldosterone (Ald), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1) and C-reactive protein (CRP), and had histologic improvement compared with model group. CONCLUSION: Shenkang Injection can improve the kidney function, decrease the levels of serum inflammatory factors,improve the oxidative status and reduce the degree of hypertensive renal damage.

DMSO-soluble cigarette smoke particles alter the expression of endothelin B receptor in rat coronary artery.

In coronary artery diseases, cigarette smoking is a risk factor and the endothelin system plays a key role in the pathogenesis. This study was to examine if dimethylsulfoxide-soluble smoke particles (DSP) upregulate endothelin type-B (ETB) receptors in the coronary artery and investigate the mechanism. The isolated rat coronary arteries were organ-cultured for 24 h. The contractile response of the coronary artery was recorded by myograph. The mRNA and protein expression of the ETB receptors was studied using quantitative real-time PCR and immunohistochemistry. Results showed that the ETB receptor agonist, sarafotoxin 6c, induced a weak contraction in the fresh coronary artery. After culture, the contraction curve mediated by ETB receptor was shifted towards the left with an increased Emax of 152 ± 12%. DSP of 0.2 and 0.4 µl/ml shifted the concentration-contractile curves towards the left with further increased Emax of 270 ± 26 and 280 ± 29%, respectively. The culture increased ETB receptor mRNA and protein levels from fresh arteries, which was further enhanced by DSP. PD98059 (ERK1/2 inhibitor), wedelolactone (NF-κB inhibitor), actinomycin D or cycloheximide significantly inhibited the DSP-enhanced contraction and expression of mRNA and protein of the ETB receptor. However, SB203580 (p38 inhibitor) further increased DSP-enhanced contraction and protein expression of the ETB receptor. The results indicate that DSP upregulates ETB receptors in rat coronary artery via ERK1/2 and the NF-κB pathway.

Lipid soluble smoke particles upregulate endothelin receptors in rat basilar artery.

The present study examines the effect of dimethylsulphoxide-soluble particles (DSP) from cigarette smoke on endothelin (ET) receptors in the basilar artery. The contractile responses to ET-1 (ET(A) and ET(B) receptors agonist) and sarafotoxin 6c (ET(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of ET receptors were determined with real-time PCR, while the protein level was evaluated by immunohistochemistry. The results showed that a DSP concentration of 0.4 microl/ml increased the contractile responses induced by sarafotoxin 6c and ET-1 and the mRNA and protein levels of the ET receptors. Inhibitor SB203580 (a p38 inhibitor), staurosporine (a PKC inhibitor) or wedelolactone (a NF-kappaB inhibitor) attenuated the elevated sarafotoxin 6c-induced contraction, the increased mRNA expression and protein levels of the ET(B) receptor induced by DSP. The effects on the ET(A) receptor induced by DSP 0.4 microl/ml were inhibited by co-incubation with PD98059 (an ERK1/2 inhibitor) or SP600125 (a JNK inhibitor) and were further enhanced by SB203580. The results indicate that DSP 0.4 microl/ml upregulates the ET(B) receptor of basilar arterial smooth muscle cells via activation of the p38 pathway and transcriptional factor NF-kappaB, while also upregulating the ET(A) receptor via activation of the ERK1/2 and JNK pathways. Additionally, the p38 pathway seems to be involved in the feedback regulation of the ET(A) receptor.

Down-regulation of alpha-adrenoceptor expression by lipid-soluble smoke particles through transcriptional factor nuclear factor-kappaB pathway.

Smoking is a strong risk factor for cardiovascular disease that is a leading cause of death and disability in Western countries. The present study was designed to investigate the effect of lipid-soluble smoke particles (DSP) on alpha-adrenoceptor expression in organ culture of rat mesenteric arteries and human epiploon arteries. Myograph and real-time reverse transcription-polymerase chain reaction were employed to assess vascular smooth muscle contractibility and the receptor mRNA expression in the smooth muscle cells. Organ culture of the arterial segments in the presence of DSP (0.2 microl/ml) resulted in a significantly decreased contractile response to norepinephrine, compared to control (i.e. in the presence of dimethyl sulfoxide) (P < 0.05). This was in parallel with a down-regulation of alpha(1A)-adrenoceptor mRNA expression in the smooth muscle, while alpha(2)-adrenoceptor mRNA expression remained unchanged. General transcription inhibitor actinomycin D (10(-5.4 )M), but not the translational inhibitor cycloheximide (10(-5 )M), significantly abolished the DSP-induced depressed contraction to norepinephrine. IMD-0354 (10(-7.5 )M), a specific nuclear factor-kappaB (NF-kappaB) pathway inhibitor, markedly reversed the DSP-induced down-regulation of alpha(1A)-adrenoceptor expression in the smooth muscle at both functional and mRNA levels. Thus, we have demonstrated that smoking-induced down-regulation of alpha(1A)-adrenoceptor expression was via the transcriptional factor NF-kappaB pathway.

Study on the Mechanism of Fibronectin Adhesion to NIH3T3 cell Stimulated by Retinoic Acid.

Retinoic acid (RA) is an inducer of cell differentiation. Recently, it was discovered that RA widely works on cells, causing a series of physiological alterations, and may make the tumor cell differentiation back to the normal cells, but little is known about its role in the adhesion of the cell to the extracellular matrix (Fn). We observed that RA increased the ability of NIH3T3 cells to adhere to Fn in a dose-dependent manner. The cell adhesion to fibronectin was promoted by 20% with 32 &mgr;mol/L RA, but not to polylysine. We use anti-integrin alpha(5)-subunit and beta(1)-subunit monoclonal antibodies to measure the amounts of integrin on the cell surface by FCM. It showed that the amounts of alpha(5) and beta(1) subunits of integrin did not change when the NIH3T3 cells were treated with RA for 24 h. But the incorporation of (3)H-mannose was increased by 53 % and those of tri- or tetra- antennary or bisecting complex type oligosaccharides were increased by 13 %. These results indicated that the increased adhesion of the cells to Fn in presence of RA may be caused by altering amounts and types of the oligosaccharides of the glycoprotein on the cell surface.