Antifungal activities and action mechanisms of compounds from Tribulus terrestris L.

Antifungal activity of natural products is being studied widely. Saponins are known to be antifungal and antibacterial. We used bioassay-guided fractionation to have isolated eight steroid saponins from Tribulus terrestris L. , which were identified as hecogenin-3-O-beta-D-glucopyranosyl （ 1→4 ）-beta-D-galactopyranoside （ TTS-8）, tigogenin-3-O-beta-D-glucopyranosyl （ 1 →4 ）-beta-D-galactopyranoside （ TTS-9 ）, hecogenin-3-O-beta-D-glucopyranosyl （ 1 → 2 ）-beta-D-glucopyranosyl （ 1 4 ）-beta-D-galactopyranoside （ TTS-10 ）, hecogenin-3-O-beta-D-xylopyranosyl （ 1→3 ）-beta-D-glucopyranosyl （ 1→ 4 ）-beta-D-galactopyranoside （ TTS-11 ）, tigogenin-3-O-beta-D-xylopyranosyl （ 1→ 2 ）-[- beta-D-xylopyranosyl （ 1 → 3 ） ]-beta-D-glucopyranosyl （1→4）-[alpha-L-rhamnopyranosyl （1→2）]-beta-D-galactopyranoside （TTS-12）, 3-O-Ebeta-D-xylopyranosyl （1→2）-[beta-D- xylopyranosyl （1→3）]-beta-D-glucopyranosyl （ 1→4）-[alpha-L-rhamnopyranosyl （ 1→2 ）]-beta-D-galactopyranosy13-26-O-beta- D-glucopyranosyl-22-methoxy-（3beta, 5alpha, 25R）-furostan-3,26-diol （TTS-13）, hecogenin-3-O-beta-D-glucopyranosyl （ 1→ 2）-[beta-D-xylopyranosyl （ 1→ 3 ）]-beta-D-glucopyranosyl （ 1 → 4 ）-beta-D-galactopyranoside （ TTS-14 ）, tigogenin-3-O-beta-D- glucopyranosyl （ 1→ 2 ）-[ beta-D-xylopyranosyl （ 1 → 3 ）]-beta-D-glucopyranosyl （ 1 → 4 ）-beta-D-galaetopyranoside （ TTS-15 ）. The in vitro antifungal activities of the eight saponins against five yeasts, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans were studied using microbroth dilution assay. In vivo activity of TTS-12 in a Candida albicans vaginal infection model was studied in particular. The results showed that TTS-12 and TTS-15 were very effective against several pathogenic candidal species and Cryptococcus neoformans in vitro. It is noteworthy that TTS-12 and TTS-15 were very active against Candida albicans （MIC50 = 10 and 2.3 microg/ml） and Cryptococcus neoformans （MIC50 = 1.7 and 6. 7 microg/ml）.     

In vitro and in vivo antifungal activities of the eight steroid saponins from Tribulus terrestris L. with potent activity against fluconazole-resistant fungal pathogens

Antifungal activity of natural products is being studied widely. Saponins are known to be antifungal and antibacterial. We have isolated eight steroid saponins from Tribulus terrestris L. , namely TTS-8, TTS-9, TTS-10, TTS-11, TTS-12, TTS-13, TTS-14 and TTS-15. TTS-12 and TTS-15 were identified as tigogenin-3-O-β-D-xylopyranosyl（1→ 2）-[-β-D-xylopyranosyl（ 1 → 3 ） 3-β- D-glucopyranosyl （ 1 → 4 ）- 1- α-L-rhamnopyranosyl （ 1 → 2 ） 3-β-D-galactopyranoside and tigogenin-3-O-β-D-glucopyranpyranosyl（1→2）-[-β-D-xylopyranosyl（1→ 3）3-β-D-glucopyranosyl（1→4）-β-D-galactopyranoside, respectively. The in vitro antifungal activities of the eight saponins against six fluconazole-resistant yeasts, Candida albicans, Candida glabrata, Candida para psilosis , Candida tropicalis , Candida krusei , and Cryptococcus neo f ormans were studied using microbroth dilution assay. The results showed that TTS-12 and TTS-15 were very effective against several pathogenic candidal species and C. neoformans in vitro. It is noteworthy that TTS-12 and TTS-15 were very active against fluconazole-resistant C. albicans （MIC80 = 4.4, 9.4 mg/ml）, C. neoformans （MIC80 =10.7, 18.7 mg/ml） and inherently resistant C. krusei （MIC80 =8.8, 18.4 mg/ml）. So in vivo activity of TTS-12 in a vaginal infection model with fluconazole-resistant C. albicans was studied in particular. Our studies revealed TTS-12 also showed in vivo activities against fluconazole-resistant yeasts. In conclusion, steroid saponins TTS-12 and TTS-15 from Tribulus terrestris L. have significant in vitro antifungal activity against fluconazole-resistant fungi, especially TTS-12 also showed in vivo activity against fluconazole-resistant C. albicans.     

Molecular Characterization of Drug-Resistant Beijing Family Isolates of Mycobacterium Tuberculosis from Tianjin,China

Objective Tuberculosis remains a severe public health issue, and the Beijing family of mycobacterium tuberculosis （M. tuberculosis） is widespread in East Asia, especially in some areas in China, like Beijing and Tianjin. This study aimed at determining the mutation patterns of drug-resistant Beijing strains of M. tuberculosis isolated from Tianjin, China. Methods A total of 822 M. tuberculosis isolates were screened for drug resistance by an absolute concentration method and the genotype was identified by PCR. 169 drug-resistant isolates of the Beijing family were analyzed for the potential mutations in the rpoB, katG, inhA promoter region and in rpsL, rrs and embB genes, which are associated with resistance to rifampin （RFP）, isoniazid （INH）, streptomycin （SM） and ethambutol （EMB） respectively by PCR and DNA sequencing. Results Fifty-eight out of 63 RFP-resistant isolates were found to carry the mutations within the 81-bp RFP resistance determining region （RRDR） of the rpoB gene and the most frequent mutations occurred at codon 531 （44.4%）, 526 （28.6%）, and 516 （7.9%） respectively. 16 mutation pattems affecting 12 different codons around the RRDR of rpoB were found. Of 116 INH-resistant isolates, 56 （48.3%） had the mutation of katG 315 （AGC→ACC） （Ser→Thr）, 3 （2.6%） carried S315N （AGC→AAC） and 27 （16.0%） had the mutation of inhA-15A→T. 84 out of 122 SM-resistant isolates （68.9%） displayed mutations at the codons 43 or 88 with AAG→AGG （Lys→Arg） of the rpsL gene and 22 （18.0%） with the mutations at positions 513A→C, 516C→T or 905 A→G in the rrs gene. Of 34 EMB-resistant isolates, 6 had mutation with M306V （ATG→GTG）, 3 with M306I （ATG→ATT）, 1 with M306I （ATG→ATA）, 1 with D328Y （GAT→TAT）, 1 with V348L （GTC→CTC）, and 1 with G406S （GGC→AGC） in the embB gene. Conelusion These novel findings extended our understanding of resistance-related mutations in the Beijing strains of M. tuberculosis and may provide a scientific basis for development of new strategies for diagnosis and control of tuberculosis in China and other countries where Beijing strains are prevalent.     

Critical Amino Acid Residues for Nicotine 5＇ -Hydroxylation in Human CYP2A Enzymes

Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by     

High Glucose Promotes the CTGF Expression in Human Mesangial Cells via Serum and Glucocorticoid-induced Kinase 1 Pathway

The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.     

Limited value of recovery phase-limited ST segment depression of treadmill exercise test

Background Clinical meaning of recovery phase limited ST segment depression of a treadmill exercise test is controversial.The aim of this study was to re-assess the diagnostic and prognostic value of ST segment depression during the recovery phase with the active phase of a treadmill exercise test in suspected coronary artery disease patients.Methods Clinical,exercise and angiographic data were retrospectively collected from 602 patients in the study.Five hundred and seventy-six patients developed ST segment depression during the active phase of the treadmill exercise test （group 1） and 26 patients developed ST segment depression only during the recovery phase （group 2）.Results With similar major clinical features,the prevalence of significant coronary artery stenosis and average Gensini scores were lower in the recovery phase-limited depression patients （group 2 vs.group 1,50.0% vs.66.9%,P=0.031 and group 2 vs.group 1,1.5 vs.8.5,P=0.04）.At a median follow up of 50.9 months for 22 group 2 and 34.8 months for 438 group 1 patients,the prevalence of total cardiac events was higher in group 1 than in group 2 patients （RR 1.60,95% Cl 1.00-2.54,P=0.049）.Conclusion The present study provides preliminary evidence that the diagnostic and prognostic value of recovery phaselimited ST segment depression of treadmill exercise test is limited.     

Metformin ameliorates insulin resistance in L6 rat skeletal muscle cells through upregulation of SIRT3

Background SIRT3 is an important regulator in cell metabolism, and recent studies have shown that it may be involved in the pharmacological effects of metformin. However, the molecular mechanisms underlying this process are unclear. Methods The effects of SIRT3 on the regulation of oxidative stress and insulin resistance in skeletal muscle were evaluated in vitro. Differentiated L6 skeletal muscle cells were treated with 750 pmol/L palmitic acid to induce insulin resistance. SIRT3 was knocked down and overexpressed in L6 cells. SIRT3, nuclear factor kappa-light-chain-enhancer of activated B cells （NF-KB） p65, c-Jun N-terminal kinase 1 （JNK1）, and superoxide dismutase 2 （SOD2） were evaluated by Western blotting. Results Over expression of SIRT3 increased glucose uptake and decreased ROS production in L6-1R cells as well as in L6 cells. Knock-down of SIRT3 induced increased production of ROS while decreased glucose uptake in both L6 and L6- IR cells, and these effects were reversed by N-acetyI-L-cysteine （NAC）. Metformin increased the expression of SIRT3 （1.5- fold） and SOD2 （2-fold） while down regulating NF-KB p65 （1.5-fold） and JNK1 （1.5-fold）. Knockdown of SIRT3 （P〈0.05） reversed the metformin-induced decreases in NF-KB p65 and JNK1 and the metformin-induced increase in SOD2 （P〈0.05）. Conclusions Upregulated SIRT3 is involved in the pharmacological mechanism by which metformin promotes glucose uptake. Additionally, SIRT3 may function as an important regulator of oxidative stress and a new alternative approach for targeting insulin resistance-related diseases.     

Value of the peripheral blood B-cells subsets in patients with ankylosing spondylitis

Background The role of B-cell remains an enigma in the pathogenesis of ankylosing spondylitis （AS）. This study aimed to investigate the distributions of B-cells and subsets in peripheral blood of AS patients and observe their changes in etanercept-treated AS patents. Methods We detected the proportions of CD19^＋ B-cell, naive B-cell （CD19^＋CD27）, memory B-cell （CD19^＋CD27dim） and plasmablast （CD19^＋CD27high） in peripheral blood of 66 patients with AS （39 at active stage, 27 at stable stage; 35 patients with peripheral joint involvement, 31 patients with axial involvement alone）, 30 patients with rheumatoid arthritis （RA） and 30 healthy volunteers using flow cytometry. And then we observed the changes of the above indexes of 39 active AS patients treated with etanercept in a randomized, double-blind, placebo-controlled trial. Results （1） Percentages of CD19^＋ B-cells in active or peripheral joint involvement AS patients increased more obviously than those in stable or axial involvement alone AS patients （both P=0.001）, and percentage of CD19^＋CD27high B-cells in AS patients with peripheral joint involvement was significantly higher than that in cases with axial involvement alone or healthy volunteers （P=0.005 and 0.006, respectively）; （2） The percentage of CD19~ B-cells in AS patients was positively correlated with Bath Ankylosing Spondylitis Disease Activity Index （BASDAI） scores, Patient＇s Global Assessment （PGA） scores, total back pain scores and nocturnal back pain scores （r=0.270, 0.255, 0.251 and 0.266, P=-0.029, 0.039, 0.042 and 0.031, respectively）; （3） At week 6 and week 12, there were no statistical differences of the percentages of B-cells and subsets between etanercept group and placebo group of AS patients （P 〉0.05）; the percentage of CD19^＋ B-cells in etanercept group was higher than that in healthy volunteers at week 12 （t=3.320, P=0.003）. Conclusions Misbalance is present in B-cells and some subsets in peripheral blood of active AS patients with peripheral joint involved. B-cells might play an important role in the pathogenesis of AS patients. The high percentage of CD19^＋ B-cells in active AS patients cannot be down-regulated after 12-week etanercept treatment.     

Association between two polymorphisms of the bone morpho- genetic protein-2 gene with genetic susceptibility to ossification of the posterior longitudinal ligament of the cervical spine and its severity

Background Ossification of the posterior longitudinal ligament （OPLL） has a strong genetic background. Previous studies have shown that bone morphogenetic protein-2 （BMP2） and BMP2 mRNA are expressed in ossifying matrix and chondrocytes adjacent to cartilaginous areas of OPLL tissues and mesenchymal cells with fibroblastic features in the immediate vicinity of the cartilaginous areas. It is suggested that BMP2 plays different roles in the different stages of development of OPLL. However, it remains unknown which factors induce ligament cells to produce BMP2. Methods OPLL patients （n=-192） and non-OPLL controls （n=304） were studied. Radiographs of the cervical spine were analyzed for extent of OPLL. We investigated whether single nucleotide polymorphisms of exons 3（-726） T/C and 3（-583） NG in the BMP2 gene are statistically associated with genetic susceptibility to OPLL in Chinese Han subjects. Results There was no statistical difference between the occurrence of exons 3（-726） T/C and 3（-583） NG and the occurrence of OPLL in the cervical spine. However, there was a significant association between occurrence of exon 3（-726） T/C polymorphism and occurrence of OPLL in males of cases and controls in the cervical spine. In addition, no significant association was found between the exons 3（-726） T/C and 3（-583） A/G with number of ossified cervical vertebrae in OPLL patients. Conclusions Exon 3（-583） A/G polymorphism in BMP2 gene is not associated with the occurrence and the extent of OPLL in the cervical spine. Chinese Han male patients with TC and CC genotypes in exon 3（-726） T/C have genetic susceptibility to OPLL but not to more extensive OPLL in the cervical spine.