Spilanthes acmella Murr. ameliorates chronic stress through improving mitochondrial function in chronic restraint stress rats.

Chronic stress is a risk factor for the development of psychiatric illnesses through impairment of the ability to appropriately regulate physiological and behavioral responses, but the molecular events that lead to damage of hippocampal neurons remain unclear. The medicinal herb Spilanthes acmella Murr. has been used as a traditional medicine for various diseases and its extracts exhibit antioxidant activity. The present study explored the molecular signals of mitochondrial dynamics and investigated the beneficial effects of S. acmella Murr. An ethyl acetate extract of this plant was used to assess mitochondrial dynamics in response to chronic restraint stress (CRS) in male Sprague-Dawley rats. The results demonstrated that the S. acmella Murr. extract reduced the expression of mitochondrial fission protein but induced HSP60, MnSOD and ATPsynthase in the hippocampus of the CRS rats. In addition, S. acmella Murr. extract reversed depressive symptoms in the forced swim test. Our findings suggested that S. acmella Murr. extract provides a potential treatment of chronic stress, and that the mechanism is associated with the alleviation of neuronal injury and maintenance of mitochondrial function.

Proteomic screening identifies the direct targets of chrysin anti-lipid depot in adipocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Overweight/obesity was mentioned by many countries as an obstacle to good health and long life, which increases risk of diseases and disorders. Previous studies suggested that the chronic low-grade inflammation present in the body was considered as the essential pathogenesis for obesity. Chrysin is extracted from traditional Chinese medicine Oroxylum indicum (Linn.) Kurz and plays a superior anti-obesity role. Chrysin could reduce the lipid depot by inhibiting the obesity-related inflammation in adipose tissue. However, the target protein for chrysin to exert its anti-obesity role are not verified. AIM OF STUDY: The present study aimed to screen and validate the target protein for chrysin to reduce the lipid depot in palmitic acid-induced 3T3-L1 adipocytes. MATERIALS AND METHODS: Obesity model was established employing 0.5 mmol/L palmitic acid-induced 3T3-L1 adipocytes through "Cocktails" method. Two-dimensional gel electrophoresis (2-DE) combined with liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the differentially expressed proteins for chrysin intervention by lipid formation in adipocytes. Gene silencing was utilized to decrease gene expression of the candidate proteins, then production of triglyceride in 3T3-L1 was detected by triglycerides assay to determine the target proteins. Ultraviolet (UV) absorption together with fluorescence spectra validated the direct target proteins of chrysin. They also computed the correlation constants of combination between chrysin and the target proteins. Molecular docking was further employed to identify the main binding amino acids between chrysin and the target protein. RESULTS: 2-DE combined with LC-MS screened four candidate proteins which were related to metabolism and inflammation. The production of triglycerides in 3T3-L1 was reduced after decreasing gene expression of Annexin A2 (ANXA2), 60 kDa heat shock protein (HSP-60) and succinyl-CoA:3-ketoacid coenzyme A transferase 1 (SCOT-S), respectively. UV spectrum showed that the absorbance spectra of ANXA2 from 260 to 300 nm shifted upwards along with the increase in chrysin concentration, meanwhile the absorbance spectra of HSP-60 from 200 to 220 nm and from 265 to 280 nm shifted slightly upwards along with the increase in chrysin concentrations. The results indicated the conjugated structures between chrysin and ANXA2 or HSP-60. Fluorescence quenching further suggested a spontaneous interaction between chrysin and ANXA2 or HSP-60. Finally, molecular docking identified the main binding amino acids between ANXA2 and chrysin were Ser22, Tyr24, Pro267, Val298, Asp299, and Lys302. CONCLUSIONS: Chrysin can reduce the amount of triglycerides by directly downregulating the inflammation-related target proteins ANXA2 and HSP-60, exerting an anti-obesity role.

HSP60 Regulates Monosodium Urate Crystal-Induced Inflammation by Activating the TLR4-NF-κB-MyD88 Signaling Pathway and Disrupting Mitochondrial Function.

Acute gout is an inflammatory response induced by monosodium urate (MSU) crystals. HSP60 is a highly conserved stress protein that acts as a cellular "danger" signal for immune reactions. In this study, we aimed to investigate the role and molecular mechanism of HSP60 in gout. HSP60 expression was detected in peripheral blood mononuclear cells (PBMCs) and plasma of gout patients. The effect and molecular mechanism of HSP60 in gout were studied in MSU crystals treatment macrophages and C57BL/6 mice. JC-1 probe and MitoSOX Red were used to measure the mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS). HSP60 expression was significantly upregulated in the PBMCs and sera of patients with acute gout (AG) compared to those with intercritical gout (IG) or healthy controls (HCs). MSU crystals induced the expression and secretion of HSP60 in the macrophages. HSP60 knockdown or overexpression affects TLR4 and MyD88 expression, IκBα degradation, and the nuclear localization of NF-κB in MSU crystal-stimulated inflammation. Further, HSP60 facilitates MMP collapse and mtROS production and activates the NLRP3 inflammasome in MSU crystal-stimulated macrophages. In MSU crystal-induced arthritis mouse models pretreated with HSP60 vivo-morpholino, paw swelling, myeloperoxidase (MPO) activity, and inflammatory cell infiltration significantly decreased. Our study reveals that MSU crystal stimulates the expression of HSP60, which accelerates the TLR4-MyD88-NF-κB signaling pathway and exacerbates mitochondrial dysfunction.

Gene expression of heat shoc kproteins/factors (HSP60, HSP70, HSP90, HSF-1, HSF-3) and antioxidant enzyme activities in heat stressed broilers treated with vitamin C.

In broiler chickens, the relationship between dietary supplementation of vitamin C and hepatic, cardiac and renal heat shock proteins (HSP60, HSP70 and HSP90), heat shock factors (HSF-1 and HSF-3) and enzymatic antioxidants requires further investigation. The current study aimed to investigate this relationship at cellular and molecular levels in a 42 days experiment. Two hundred, one-day-old broiler chicks (Ross 308) were allocated into four equal groups. Chicks in the first and third groups were thermo-neutral (TN; 22°C for 24 hours/day) and fed basal diet without or with vitamin C (1g/kg basal diet), respectively. Chicks in the second and fourth groups were heat stressed (HS; 34°C for 8 hours/day) and fed basal diet without or with vitamin C, respectively. Performance parameters were recorded throughout the experiment. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPX), Catalase (CAT) and gene expression of heat shock proteins (HSP60, 70 and 90) and heat shock factors (HSF 1 and 3) were analyzed in liver, heart and kidney tissues of the studied groups. Heat stress induced a negative impact on performance parameters, significant reduction in activities of all examined antioxidant enzymes and a significant up-regulation in heat shock proteins and factors genes in all studied tissues. Dietary supplementation of vitamin C corrected these parameters towards the normal control values. Conclusively, dietary supplementation of the examined dose of vitamin C was efficient at ameliorating the detrimental effects of heat stress on liver, heart and kidney tissues of broilers chickens at cellular and molecular levels.

Modulatory effects of curcumin on heat shock proteins in cancer: A promising therapeutic approach.

Cancer metastasis represents a multistep process, including alteration of cell adhesion/motility in the microenvironment and sustained angiogenesis, which is essential for supporting cancer growth in tissues that are distant from the primary tumor. There is growing evidence suggesting that heat shock proteins (HSPs) (also known as heat stress proteins), which constitute a family of stress-inducible proteins, may be involved in the pathogenesis of cancer. Curcumin (diferuloylmethane) is a potent anti-inflammatory, antioxidant, antimicrobial, and antitumor agent. Curcumin has been shown to regulate different members of HSPs including HSP27, HSP40, HSP60, HSP70, and HSP90 in cancer. Here, we present extent findings suggesting that curcumin may act as a potential therapeutic agent for the treatment of cancer through its regulation of HSPs.

Selenium deficiency induced an inflammatory response by the HSP60 - TLR2-MAPKs signalling pathway in the liver of carp.

Selenium (Se) is one of the essential trace elements for immune regulation and antioxidant systems in fish growth. The dietary Se plays an important role in immune regulation and inflammation by regulating HSPs and TLRs in liver of many animals. The liver is an important digestive organ in carp. Liver damage can seriously affect the growth and survival of carp. This study was conducted to determine whether Se regulated liver inflammation by affecting HSPs-TLR2 signalling and the potential mechanisms of action in common carp. The gene was analysed by qPCR. The proteins of inflammatory factors were detected by ELISA. The others proteins were analysed by Western blot. The results indicated the Se concentrations in blood and liver tissues were significantly influenced by dietary Se. The Se deficiency increased the expression of HSP60 and TLR2 and the secretion of the proinflammatory factor TNF-α, IL-1ß and IL-6, induced a low secretion of the anti-inflammatory TGF-ß, but the Se supplements could transform these events. Further research showed that with the dose-dependently decrease of Se, the HSP60 expressions were increased, and the MAPKs pathway were significantly activated by the phosphorylation of p38, JNK and ERK in liver tissue and cell. The results provide evidence that Se deficiency induced and exacerbated inflammatory injury to the liver through the HSP60 and TLR2-MAPKs signalling pathways in carp.

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides.

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.

The Antagonistic Effect of Selenium on Lead-Induced Immune Dysfunction via Recovery of Cytokine and Heat Shock Protein Expression in Chicken Neutrophils.

Lead (Pb) is a ubiquitous and toxic heavy metal and it can damage the immune system in humans and animals. Many researchers have reported that Selenium (Se) could possess various pharmacological effects in mammals. However, few studies have been carried out to investigate the protective role of Se in birds, especially in chickens. In this study, we investigated the protective effects of Se against Pb-induced inflammatory responses and the expression of heat shock proteins (HSPs) in peripheral blood neutrophils. One hundred eighty Hy-Line brown chickens were randomly divided into the control group (Con group), Se supplementation group (+Se group), Pb supplementation group (+Pb group), and the Se and Pb compound group (Se+Pb group). On the 90th day of the experiment, the peripheral blood was collected to extract neutrophils, and then, the levels of HSPs and cytokines were examined. The results showed that, after Pb treatment, the levels of IL-(1ß, 1R, 4, 8, 10, and 12ß), TGF-ß4, and HSP (27, 40, 60, 70, and 90) mRNA were significantly increased and levels of IL-2 and IFN-γ mRNA were decreased compared with those in the control group. Compared with the control group, the protein levels of HSP60 and HSP70 were also increased in the Pb treatment group. Co-administration of Se (1 mg/kg/day) and Pb resulted in a reversal of the Pb-induced cytokine changes in neutrophils accompanied by a significant decrease in HSPs. Our study demonstrated that Pb could decrease the immune function via changing the expression of cytokines and HSPs in chicken neutrophils, but Se could relieve the toxic effect induced by Pb.

Himatanthus drasticus (Apocynaceae) latex reduces oxidative stress and modulates CD4+, CD8+, FoxP3+ and HSP-60+ expressions in Sarcoma 180-bearing mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, latex of Himatanthus drasticus is used to treat inflammation, wound healing and cancer. The present study evaluated the antitumoral potential of H. drasticus latex (HdCL) in Sarcoma 180-bearing mice (S180). MATERIALS AND METHODS: HdCL was obtained in Crato-CE, Brazil. Qualitative phytochemicals assays, nuclear magnetic resonance (NMR) and microbiological analyzes were performed. Swiss mice were divided into six groups, according to tumor forms: 1) ascitic model, GI (Control; 0.9% saline), GII (S180asc) and GIII (S180asc/HdCL/14 days); 2) solid model, GIV (Control; 0.9% saline), GV (S180sol) and GVI (S180sol/HdCL/10 days). HdCL and 0.9% saline were administered at 0.2 mL, SID, by gavage, for 10 or 14 days. For ascitic model, 0.5 mL of S180 suspension (4×106 cells/mL) was inoculated intraperitoneally and for solid model, cells were inoculated subcutaneously (25 µL) on the right hind paw of mice. Blood samples were collected for hematological and oxidative stress evaluation. Thickness, volume and weight of paws were measured in solid model. After euthanasia, spleen, liver and kidney were collected in order to assess the relative organ weight. Tissue fragments of paws and popliteal lymph nodes (PLN) were analyzed by H&E and CD4+, CD8+, HSP-60+ and Foxp3+ immunohistochemistry. RESULTS: HdCL presented milky aspect and pinkish supernatant. Phenols, flavonols, flavanones, free steroids and cinnamoyl derivatives of lupeol, α-amyrin and ß-amyrin were detected at the phytochemistry analysis. HdCL did not alter the relative weight of organs, hematological parameters and volume of ascitic fluid recovered. In solid model, HdCL reduced (P < 0.05) paw volume, but did not altered thickness, paw weight and histological parameters. S180sol induced necrosis, metastasis and destruction of bone, cartilage and muscles. Bleeding, vessel congestion and oncocytes were observed in PLN. In paw, HdCL did not alter FoxP3+ and HSP-60+ expressions but reduced the CD4+ and CD8+ expressions, while at PLN, HdCL reduced the expressions of all markers. HdCL decreased (P < 0.05) serum levels of malondialdehyde in ascitic model. CONCLUSIONS: Treatment with HdCL reduced oxidative damage and modulated the expressions of CD4+, CD8+, FoxP3+and HSP-60+ in S180 solid tumor model, which can be associated to the presence of triterpenes, such as α-amyrin, ß-amyrin and lupeol cinnamate. Present data emphasizes the importance of immune system in cancer and highlights the evaluation of the pharmacological properties of plants used by population as phytoterapics.

Comparative proteomics-network analysis of proteins responsible for ursolic acid-induced cytotoxicity in colorectal cancer cells.

Ursolic acid is a key active compound present in many medicinal herbs that have been widely used in traditional Chinese medicine for the clinical treatment of various cancers. However, the precise mechanisms of its antitumor activity have been poorly understood. To identify the cellular targets of ursolic acid, two-dimensional gel electrophoresis combined with mass spectrometry was performed in this study, which identified 15 proteins with significantly altered levels in protein expression. This demonstrated that ursolic acid-induced cytotoxicity in colorectal cancer cells involves dysregulation in protein folding, signal transduction, cell proliferation, cell cycle, and apoptosis. Corresponding protein regulation was also confirmed by Western blotting. Furthermore, the study of functional association between these 15 proteins revealed that 10 were closely related in a protein-protein interaction network, whereby the proteins either had a direct interaction with each other or were associated via only one intermediary protein. In this instance, the ATP5B/CALR/HSP90B1/HSPB1/HSPD1-signaling network was revealed as the predominant target which was associated with the majority of the observed protein-protein interactions. As a result, the identified targets may be useful in explaining the anticancer mechanisms of ursolic acid and as potential targets for colorectal cancer therapy.

Role of calcium in the mitigation of heat stress in the cyanobacterium Anabaena PCC 7120.

The effects of exogenously added CaCl2 (0.25mM) on photopigments, photosynthetic O2-evolution, antioxidative enzyme activity, membrane damage, expression of two heat shock genes (groEL and groES) and apoptotic features in Anabaena 7120 under heat stress (45°C) for up to 24h were investigated. Heat stress lowered the level of photopigments; however, Ca2+--supplemented cultures showed a low level reduction in Chl a but induced accumulation of carotenoids and phycocyanin under heat stress. Photosynthetic O2-evolving capacity was maintained at a higher level in cells from Ca2+-supplemented medium. Among the antioxidative enzymes, superoxide dismutase activity was unaffected by the presence or absence of Ca2+ in contrast to increases in catalase, ascorbate peroxidase and glutathione reductase activities in cells grown in Ca2+-supplemented medium. Lower levels of lipid peroxidation were recorded in Anabaena cells grown in Ca2+-supplemented medium in comparison to cells from Ca2+--deprived medium. Target cells grown in Ca2+-deprived medium developed apoptotic features in the early stages of heat shock, while Ca2+ application seemed to interfere with apoptosis because only a few cells showed such features after 24 h of heat exposure, indicating a role for Ca2+ in maintaining cell viability under heat stress. There was also continuous up regulation of two important heat shock genes (groEL and groES) in Ca2+-supplemented cultures, exposed to heat shock, again indicating a role for Ca2+ in stress management.

Postcesarean section wound infection caused by Mycobacterium massiliense.

BACKGROUND/PURPOSE: Mycobacterium abscessus subsp. massiliense (a subspecies of the M. abscessus complex) is a rare causative agent of surgical site infection after cesarean section (C section). We tried to seek the common source of infection and unravel the optimal treatment modalities. METHODS: From September 2009 to October 2012, four postpartum women developed C-section wound infections caused by M. massiliense. Speciation of the four isolates was identified using of hsp65, rpoB, and secA1 partial gene sequencing and the Basic Local Alignment Search Tool. The erm(41) and rrl genes were detected for the possibility of inducible macrolide resistance. Pulsed-field gel electrophoresis was used as a tool of molecular epidemiology. All patients underwent intensive intravenous and oral antimycobacterial regimens. Of these patients, three underwent debridement at least once. RESULTS: All four isolates were identified as M. abscessus subsp. massiliense. All of the isolates harbored a truncated erm(41) gene without rrl gene mutations, which explains the susceptibility to clarithromycin and azithromycin. Three isolates were indistinguishable by DNA strain typing, and the fourth strain was clonal with the other three strains. Their infections were not improved in spite of teicoplanin treatment initially. These patients underwent antimycobacterial regimens with/without surgery and were all cured. DISCUSSION: Teicoplanin treatment failure, painful cutaneous nodules, and persistent wound drainage alerted us to the possibility of nontuberculous mycobacterial skin and soft tissue infection. Accurate identification of subspecies, detection of drug resistance genes, susceptibility testing, and optimal antimycobacterial agents with/without surgical debridement are warranted for successful treatment.

Anti-breast tumor activity of Eclipta extract in-vitro and in-vivo: novel evidence of endoplasmic reticulum specific localization of Hsp60 during apoptosis.

Major challenges for current therapeutic strategies against breast cancer are associated with drug-induced toxicities. Considering the immense potential of bioactive phytochemicals to deliver non-toxic, efficient anti-cancer therapeutics, we performed bio-guided fractionation of Eclipta alba extract and discovered that particularly the chloroform fraction of Eclipta alba (CFEA) is selectively inducing cytotoxicity to breast cancer cells over non-tumorigenic breast epithelial cells. Our unbiased mechanistic hunt revealed that CFEA specifically activates the intrinsic apoptotic pathway by disrupting the mitochondrial membrane potential, upregulating Hsp60 and downregulating the expression of anti-apoptotic protein XIAP. By utilizing Hsp60 specific siRNA, we identified a novel pro-apoptotic role of Hsp60 and uncovered that following CFEA treatment, upregulated Hsp60 is localized in the endoplasmic reticulum (ER). To our knowledge, this is the first evidence of ER specific localization of Hsp60 during cancer cell apoptosis. Further, our LC-MS approach identified that luteolin is mainly attributed for its anti-cancer activities. Moreover, oral administration of CFEA not only offers potential anti-breast cancer effects in-vivo but also mitigates tumor induced hepato-renal toxicity. Together, our studies offer novel mechanistic insight into the CFEA mediated inhibition of breast cancer and may potentially open up new avenues for further translational research.

Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model.

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg(-1)·day(-1) by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH.

Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

Leptin regulation of Hsp60 impacts hypothalamic insulin signaling.

Type 2 diabetes is characterized by insulin resistance and mitochondrial dysfunction in classical target tissues such as muscle, fat, and liver. Using a murine model of type 2 diabetes, we show that there is hypothalamic insulin resistance and mitochondrial dysfunction due to downregulation of the mitochondrial chaperone HSP60. HSP60 reduction in obese, diabetic mice was due to a lack of proper leptin signaling and was restored by leptin treatment. Knockdown of Hsp60 in a mouse hypothalamic cell line mimicked the mitochondrial dysfunction observed in diabetic mice and resulted in increased ROS production and insulin resistance, a phenotype that was reversed with antioxidant treatment. Mice with a heterozygous deletion of Hsp60 exhibited mitochondrial dysfunction and hypothalamic insulin resistance. Targeted acute downregulation of Hsp60 in the hypothalamus also induced insulin resistance, indicating that mitochondrial dysfunction can cause insulin resistance in the hypothalamus. Importantly, type 2 diabetic patients exhibited decreased expression of HSP60 in the brain, indicating that this mechanism is relevant to human disease. These data indicate that leptin plays an important role in mitochondrial function and insulin sensitivity in the hypothalamus by regulating HSP60. Moreover, leptin/insulin crosstalk in the hypothalamus impacts energy homeostasis in obesity and insulin-resistant states.

Effect of moxibustion treatment on cell apoptosis and expressions of heat shock protein and second mitochondrial activator of caspase in acute gastric mucosal lesion of rats.

OBJECTIVE: To investigate the effect of moxibustion-acupoint treatment with acupoints of Zusanli (ST 36) and Zhongwan (RN 12) on cell apoptosis and the expressions of heat shock protein (HSP) 60, HSP70 and second mitochondrial activator of caspase (Smac) in rat models of acute gastric mucosal lesion (AGML), and explore the mechanisms underlying protection of gastric mucosal lesion. METHODS: Twenty-four Sprague Dawley rats were divided into 3 groups, blank controlled group (group A), controlled-point group (group B) and acupoint group (group C), 8 for each. After 8-day moxibustion treatment in group B and C, gastric lavage of anhydrous ethanol was used to created AGML in all three groups. The Guth method was employed to measure the ulcer index (UI) of gastric mucosal lesion and immunohistochemistry used to measure apoptosis with apoptosis index (AI) and examine the expressions of HSP60, HSP70 and Smac. RESULTS: Compared with group A, the expressions of UI, AI, Smac and HSP60 were markedly elevated in group B (P < 0.05 or P < 0.01). However the expression of HSP70 showed no obvious change (P > 0.05); the expressions of UI, HSP60 and HSP70 were markedly elevated in group C (P < 0.01) while those of AI and Smac became obviously suppressed (P < 0.01). Compared with group B, the expressions of UI, AI and Smac decreased significantly in group C (P < 0.01) while those of HSP60 and HSP70 increased markedly (P < 0.01), and the expressions of HSP60 and HSP70 were considerably up-regulated (P < 0.01). CONCLUSION: The moxibustion treatment could alleviate the gastric mucosal lesion caused by anhydrous ethanol, induce the over-expressions of HSP60 and HSP70, and down-regulate the expression of Smac, which could suppress cell apoptosis.

Rescue of PINK1 protein null-specific mitochondrial complex IV deficits by ginsenoside Re activation of nitric oxide signaling.

PINK1, linked to familial Parkinson's disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for new therapeutics targeting mitochondrial dysfunction in Parkinson's disease.

Heat shock protein 60 of filarial parasite Brugia malayi: cDNA cloning, expression, purification and in silico modeling and analysis of its ATP binding site.

We report here cloning and expression of full length mitochondrial HSP60 gene of Brugia malayi adult worm (mtHSP60bm), purification of the gene product by affinity chromatography, its in silico 3D structure and the sequence homology of the protein with Escherichia coli GroEL/ES and human HSP60. The ATP binding pocket of human HSP60 and mtHSP60bm were analyzed and compared using in silico models. The distribution of HSP60 in different life-stages of the parasite was determined using antibodies raised against recombinant mtHSP60bm (rmtHSP60bm). mtHSP60bm was present in all life-stages of the parasite except third stage infective larvae, in which it could be induced by heat-shock, and showed high degree of homology with E. coli GroEL/ES. The ATP binding pocket of HSP60 in humans, E. coli and B. malayi were also found structurally conserved. This similarity between human and mtHSP60bm might be useful in understanding the host-parasite interactions. This is the first ever report on distribution, cloning, sequence homology and ATP binding site of mtHSP60bm.

Immunotherapy for TB.

Mycobacterium tuberculosis was one of the first human pathogens to be identified as the cause of a specific disease--TB. TB was also one of the first specific diseases for which immunotherapy was attempted. In more than a century since, multiple different immunotherapies have been attempted, alongside vaccination and antibiotic treatment, with varying degrees of success. Despite this, TB remains a major worldwide health problem that causes nearly 2 million deaths annually and has infected an estimated 2 billion people. A major reason for this is that M. tuberculosis is an ancient human pathogen that has evolved complex strategies for persistence in the human host. It has thus been long understood that, to effectively control TB, we will need to address the ability of the pathogen to establish a persistent, latent infection in most infected individuals. This review discusses what is presently known about the interaction of M. tuberculosis with the immune system, and how this knowledge has been used to design immunotherapeutic strategies.