Guaianolides from Saussurea affinis

Cynaropicrin, 11βH-11,13-dihydrodesacylcynaropicrin, aguerins A and B, isoamberboin and the new guaianolides saussureolide and 11βH-11,13-dihydrodesacylcynaropicrin 8-β-d-glucoside were isolated from Saussurea affinis.     

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

     

MUC4 mucin potentiates pancreatic tumor cell proliferation, survival, and invasive properties and interferes with its interaction to extracellular matrix proteins

MUC4, a transmembrane mucin, is aberrantly expressed in pancreatic adenocarcinomas while remaining undetectable in the normal pancreas. Recent studies have shown that the expression of MUC4 is associated with the progression of pancreatic cancer and is inversely correlated with the prognosis of pancreatic cancer patients. In the present study, we have examined the phenotypic and molecular consequences of MUC4 silencing with an aim of establishing the mechanistic basis for its observed role in the pathogenesis of pancreatic cancer. The silencing of MUC4 expression was achieved by stable expression of a MUC4-specific short hairpin RNA in CD18/HPAF, a highly metastatic pancreatic adenocarcinoma cell line. A significant decrease in MUC4 expression was detected in MUC4-knockdown (CD18/HPAF-siMUC4) cells compared with the parental and scrambled short interfering RNA-transfected (CD18/HPAF-Scr) control cells by immunoblot analysis and immunofluorescence confocal microscopy. Consistent with our previous observation, inhibition of MUC4 expression restrained the pancreatic tumor cell growth and metastasis as shown in an orthotopic mouse model. Our in vitro studies revealed that MUC4-associated increase in tumor cell growth resulted from both the enhanced proliferation and reduced cell death. Furthermore, MUC4 expression was also associated with significantly increased invasiveness (P < or = 0.05) and changes in actin organization. The presence of MUC4 on the cell surface was shown to interfere with the tumor cell-extracellular matrix interactions, in part, by inhibiting the integrin-mediated cell adhesion. An altered expression of growth- and metastasis-associated genes (LI-cadherin, CEACAM6, RAC1, AnnexinA1, thrombomodulin, epiregulin, S100A4, TP53, TP53BP, caspase-2, caspase-3, caspase-7, plakoglobin, and neuregulin-2) was also observed as a consequence of the silencing of MUC4. In conclusion, our study provides experimental evidence that supports the functional significance of MUC4 in pancreatic cancer progression and indicates a novel role for MUC4 in cancer cell signaling.     

Evaluation of Hs-CRP Levels and Interleukin 18 (-137G/C) Promoter Polymorphism in Risk Prediction of Coronary Artery Disease in First Degree Relatives

Background

Coronary Artery Disease (CAD) is clearly a multifactorial disease that develops from childhood and ultimately leads to death. Several reports revealed having a First Degree Relatives (FDRS) with premature CAD is a significant autonomous risk factor for CAD development. C - reactive protein (CRP) is a member of the pentraxin family and is the most widely studied proinflammatory biomarker. IL-18 is a pleiotrophic and proinflammatory cytokine which is produced mainly by macrophages and plays an important role in the inflammatory cascade.

Methods and Results

Hs-CRP levels were estimated by ELISA and Genotyping of IL-18 gene variant located on promoter -137 (G/C) by Allele specific PCR in blood samples of 300 CAD patients and 300 controls and 100 FDRS. Promoter Binding sites and Protein interacting partners were identified by Alibaba 2.1 and Genemania online tools respectively. Hs-CRP levels were significantly high in CAD patients followed by FDRS when compared to controls. In IL-18 -137 (G/C) polymorphism homozygous GG is significantly associated with occurrence of CAD and Hs-CRP levels were significantly higher in GG genotype subjects when compared to GC and CC. IL-18 was found to be interacting with 100 protein interactants.

Conclusion

Our results indicate that Hs-CRP levels and IL-18-137(G/C) polymorphism may help to identify risk of future events of CAD in asymptomatic healthy FDRS.     

Luminescence studies of perturbation of tryptophan residues of tubulin in the complexes of tubulin with colchicine and colchicine analogues

Tubulin, a heterodimeric (alphabeta) protein, the main constituent of microtubules, binds efficiently with colchicine (consisting of a trimethoxybenzene ring, a seven-member ring and methoxy tropone moiety) and its analogues, viz., demecolcine and AC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone]. Tubulin contains eight tryptophan (Trp) residues at A21, A346, A388, A407, B21, B103, B346, and B407 in the two subunits. The role of these eight Trp residues in this interaction and also their perturbation due to binding have been explored via time-resolved fluorescence at room temperature and low-temperature (77 K) phosphorescence in a suitable cryosolvent. Both the time-resolved fluorescence data and 77 K phosphorescence spectra indicate that the emitting residues move toward a more hydrophobic and less polar environment after complex formation. The environment of emitting Trps in the complex also becomes slightly more heterogeneous. Our analysis using the experimental results, the calculation of the accessible surface area (ASA) of all the Trps in the wild type and tubulin-colchicine complex [Ravelli, R. B. G., et al. (2004) Nature 428, 198-202], the distance of the Trp residues from the different moieties of the colchicine molecule, the knowledge of the nature of the immediate residues (<5 A) present near each Trp residue, and the calculation of the intramolecular Trp-Trp energy transfer efficiencies indicate that Trp A346, Trp A407, Trp B21, and Trp B407 are the major contributors to the emission in the free protein, while Trp B21 and Trp B103 are mainly responsible for the emission of the complexes. A comparative account of the photophysical aspects of the drug molecules bound to protein in aqueous buffer and in buffer containing 40% ethylene glycol has been presented. The quantum yield and average lifetime of fluorescence in tubulin and its complexes with colchicine are used to predict the possible donors and the energy transfer (ET) efficiency in the ET process from Trps to colchicine in the complex. This study is a unique attempt to identify the Trp residues contributing to the emission in the free protein and in a complex of a multi-Trp protein with a drug molecule without performing the mutation of the protein.     

Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.     

The Hha–TomB toxin–antitoxin module in Salmonella enterica serovar Typhimurium limits its intracellular survival profile and regulates host immune response

Cell Biology and Toxicology - The key to bacterial virulence relies on an exquisite balance of signals between microbe and hosts. Bacterial toxin–antitoxin (TA) system is known to play a...     

Characterization of an oxidoreductase from the arylamine N-acetyltransferase operon in Mycobacterium smegmatis

Mycobacterium?tuberculosis, the most successful bacterial pathogen, causes tuberculosis, a disease that still causes more than 2 million deaths per year. Arylamine N-acetyltransferase is an enzyme that is conserved in most Mycobacterium spp. The nat gene belongs to an operon that is important for the intracellular survival of M. tuberculosis within macrophages. The nat operon in Mycobacterium smegmatis and other fast-growing mycobacterial species has a unique organization containing genes with uncharacterized function. Here, we describe the biochemical, biophysical and structural characterization of the MSMEG_0308 gene product (MS0308) of the M. smegmatis nat operon. While characterizing the function of MS0308, we validated the oxidoreductase property; however, we found that the enzyme was not utilizing dihydrofolate as its substrate, hence we first report that MS0308 is not a dihydrofolate reductase, as annotated in the genome. The structure of this oxidoreductase was solved at 2.0 ? in complex with the cofactor NADPH and has revealed the hydrophobic pocket where the endogenous substrate binds.