Anti-Obesity and Lipid Lowering Activity of Bauhiniastatin-1 is Mediated Through PPAR-γ/AMPK Expressions in Diet-Induced Obese Rat Model.

Objective: To evaluate the therapeutic efficacy and underlying molecular mechanisms of Bauhiniastatin-1 (BSTN1) to alleviate adiposity in diet-induced obese rodent model and in 3T3-L1 cells. Methods: BSTN1 was purified and confirmed through HPLC. In-vitro experiments such as MTT assay, Oil Red-O (ORO) stain, cellular lipid content, glycerol release and RT-PCR analysis were performed in 3T3-L1 cells in the presence and absence of BSTN1. In animal experiments, rats were divided into Group-I: normal pellet diet-fed, Group-II: HFD-fed, Groups-III, IV and V: HFD-fed BSTN1 (1.25, 2.5, and 5 mg/kg.b.wt./day/rat)-treated and Group-VI: HFD-fed Orlistat-treated. The rats were fed either normal diet or high fat diet (HFD) for 18 weeks and water ad-libitum. BSTN1 was orally administered from 13th week onwards to the selected HFD-fed groups. Body composition parameters, biochemical assays, histopathology examination and western blot analysis were performed to identify the predicted targets related to obesity. Molecular docking studies threw light on the binding interactions of BSTN1 against PPAR-γ, FAS and AMPK. Results: BSTN1 at 20 µM significantly (p < 0.001) inhibited adipocyte differentiation and lipid accumulation in 3T3-L1 cells. A conspicuous down-regulation in the mRNA expression levels of PPAR-γ, FAS and SREBP1 was observed but AMPK expression remained unchanged in BSTN1 treated 3T3-L1 cells. A substantial decrease in body weight gain, fat percent, total body fat, serum and liver lipid profile (except high-density lipoprotein), glucose, insulin and insulin resistance in BSTN1 treated rats was noticed in a dose dependent manner. In BSTN1 (5 mg/kg.b.wt.)-treated groups significantly (p < 0.01) elevated plasma adiponectin level but reduced leptin level as well as fall in serum AST and ALT were noticed. Further, the disturbed structural integrity and architecture of adipose and hepatic tissues due to high fat diet feeding were considerably recovered with BSTN1 treatment. Down-regulation in the protein expression level of PPAR-γ and activation of AMPK through phosphorylation was observed in BSTN1 treated rats than the untreated. Molecular docking studies revealed strong binding interactions of BSTN1 against PPAR-γ and AMPK and thus supported the experimental results. Conclusion: Taken together, the results suggest that BSTN1 could be a promising pharmacological molecule in the treatment of obesity and dyslipidemia.

Pterocarpus santalinus L. extract mitigates gamma radiation-inflicted derangements in BALB/c mice by Nrf2 upregulation.

Plant-based natural extracts contain several nutrients and bioactive compounds, such as phenolics and flavonoids, that possess various health-promoting activities. This study investigated the effects of polyphenols from Pterocarpus santalinus hydroalcoholic extract (PSHE) against gamma radiation-induced derangements via the upregulation of Nrf2. Ultra High Performance Liquid Chromatography Coupled to High Resolution Mass Spectrometry (UHPLC-HRMS/MS) analysis was performed to identify the possible radioprotectors. In vivo and in vitro studies, namely Real-Time-PCR (RT-PCR) analysis, Reactive Oxygen Species (ROS) scavenging activity, lipid peroxidation and GSH levels, DNA damage and cell death studies, anti-inflammatory (Sandwich ELISA), immunomodulatory studies (antibody staining), and model free radical scavenging assays, were performed. Vanillic acid, protocatechuic acid, para-hydroxybenzoic acid, chlorogenic acid, TNF-α inhibitor (Eudesmin), isoflavone (Daidzein 7-o-glucoside), astragalin (Kaempferol 3-o-glycoside), and other polyphenols were identified in PSHE using UHPLC-HRMS/MS analysis. Prophylactic administration of PSHE (-1 h) rendered more than 33% survival in mice exposed to 8 Gy whole-body-irradiation with increased mice survival and recovery of bone marrow and spleen cellularity. Real-time RT-PCR analysis showed that PSHE treatment (50 µg/mL) upregulated Nrf2, HO-1, and GPX-1 in mice splenocytes. At 50 µg/mL, PSHE reduced ROSscavenging activity, mitochondrial and spleen membrane lipid peroxidation levels, DNA damage, and cell death, and increased GSH levels. At 10 µg/mL, PSHE treatment diminished the content of IL-6 and TNF-α. At 50 µg/mL, PSHE suppressed lymphocyte proliferation. These findings indicate that polyphenols of PSHE possess marked antioxidant, anti-inflammatory, and immunomodulatory capacities, which play important roles in the prevention of radiation damage.

A novel anti-NS2BNS3pro antibody-based indirect ELISA test for the diagnosis of dengue virus infections.

Dengue virus reportedly circulates as four genetically distinct serotypes for which there is no widely accepted vaccine or drug at present. Morbidity and mortality caused by this virus are alarming for the possible increased threat to human health. A suitable diagnostic test is the prerequisite for designing and developing control measures. But, the tests being employed at present possess one or the other drawback for this disease diagnosis. During the dengue virus infections, NS2B is essential for the stability and catalytic activity of the NS3 protease. N-terminal 185 amino acids of NS3 protease domain along with hydrophilic portion of NS2B (NS2BNS3pro) is being used to screen dengue inhibitors but not for diagnosis until now. In the present study, we have used purified NS2BNS3pro as an antigen to trap anti-NS2BNS3pro antibodies of the clinical samples. Antibodies were detected successfully in both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. In ELISA, antibodies were detected in both primary and secondary infections of all serotypes. Interestingly, 17 samples declared as other febrile infections by NS1 and IgM/IgG tests were found to be positive in present test, which were further confirmed by reverse-transcription polymerase chain reaction. In silico studies suggested the absence of conserved epitopes between NS2BNS3pro and the counterpart in JEV, Zika, and CHIKV, indicating less possibility of crossreaction, which was in turn confirmed by using synthetic peptides representing the above epitopes. Statistical analysis with 76% specificity, 87% sensitivity, and 95% concordance also supported the present test as a suitable test for large scale diagnosis of dengue virus infections.

Structure of the catalytic domain of the Clostridium thermocellum cellulase CelT.

Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1 Šresolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed ß-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.

Dimerization is important for the GTPase activity of chloroplast translocon components atToc33 and psToc159.

Arabidopsis Toc33 (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg(130)) has been implicated in the formation of the atToc33 dimer and in intermolecular GTPase activation within the dimer. Here we report the crystal structure at 3.2-A resolution of an atToc33 mutant, atToc33(R130A), in which Arg(130) was mutated to alanine. Both in solution and in crystals, atToc33(R130A) was present in its monomeric form. In contrast, both wild-type atToc33 and another pea Toc GTPase homologue, pea Toc159 (psToc159), were able to form dimers in solution. Dimeric atToc33 and psToc159 had significantly higher GTPase activity than monomeric atToc33, psToc159, and atToc33(R130A). Molecular modeling using the structures of psToc34 and atToc33(R130A) suggests that, in an architectural dimer of atToc33, Arg(130) from one monomer interacts with the beta-phosphate of GDP and several other amino acids of the other monomer. These results indicate that Arg(130) is critical for dimer formation, which is itself important for GTPase activity. Activation of GTPase activity by dimer formation is likely to be a critical regulatory step in protein import into chloroplasts.

Toc GTPases.

Chloroplasts import more than 90% of their protein constituents from the cytosol. The import is mediated by translocon complexes located in the chloroplast envelope and the stroma. This review focuses on the two GTPases in the Toc (translocon at the outer envelope membrane of chloroplasts) complex. Hypotheses are presented about gating across the outer membrane and the possible functional states of the GTPases.

Effect of dimethyl sulphoxide on the crystal structure of porcine pepsin.

The structure of porcine pepsin crystallized in the presence of dimethyl sulphoxide has been analysed by X-ray crystallography to obtain insights into the structural events that occur at the onset of chemical denaturation of proteins. The results show that one dimethyl sulphoxide molecule occupies a site on the surface of pepsin interacting with two of its residues. An increase in the average temperature factor of pepsin in the presence of dimethyl sulphoxide has been observed indicating protein destabilization induced by the denaturant. Significant increase in the temperature factor and weakening of the electron density have been observed for the catalytic water molecule located between the active aspartates. The conformation of pepsin remains unchanged in the crystal structure. However, the enzyme assay and circular dichroism studies indicate that dimethyl sulphoxide causes a slight change in the secondary structure and complete loss of activity of pepsin in solution.

Purification, crystallization and preliminary X-ray diffraction analysis of the catalytic domain of adenylyl cyclase Rv1625c from Mycobacterium tuberculosis.

The Rv1625c gene product is an adenylyl cyclase identified in the genome of Mycobacterium tuberculosis strain H37Rv. It shows sequence similarity to the mammalian nucleotide cyclases and functions as a homodimer, with two substrate-binding sites at the dimer interface. A mutant form of the catalytic domain of this enzyme, K296E/F363R/D365C (KFD-->ERC), was overexpressed in Escherichia coli cells in a soluble form. Crystals were obtained using the hanging-drop vapour-diffusion method with PEG 8000 as a precipitant. The protein crystallized in space group P4(1), with unit-cell parameters a = b = 71.25, c = 44.51 A. X-ray diffraction data were collected to a resolution of 3.4 A and the structure has been solved by the molecular-replacement method using a previously built theoretical model of the protein as the search molecule.