Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

Conceived and designed the experiments: FS PC BC ES AM. Performed the experiments: FS RS ES PF SR. Analyzed the data: FS BC ES PF GEK PFC AM. Contributed reagents/materials/analysis tools: PC PB GC. Wrote the paper: FS GEK BC AM.The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.Yeshttp://www.plosone.org/static/editorial#pee

Role of an Estrogen-Upregulated 64.0-kDa Uterine Fluid Glycoprotein in Improving Fertility in Women

To evaluate the significance of an estrogen-upregulated 64.0-kDa human uterine fluid (hUF) glycoprotein in relation to promotion of fertility. Prospective study. Department of Assisted Reproductive Biology Unit, Institute of Reproductive Medicine, Kolkata. India. Sixty-three women with unexplained infertility having normal ovulatory cycle with normal endocrine profile and 18 parous women. Women either received no stimulation (n � 35) or were stimulated with clomiphene citrate (CC) alone (100 mg/day from day 3 to day 7; n � 56) or in combination with pure FSH (75 IU on day 3 and day 8; n � 54) and were subjected to IUI. Parous women (n � 18) receiving no treatment served as control and were followed-up for spontaneous ovulation. Main Outcome Measure(s): Uterine fluid protein profile, relative intensity of 64.0-kDa protein, number of mature follicles, endometrial thickness, and pregnancy. Expression of the 64.0-kDa protein exhibited positive correlation with prevailing estradiol levels, but the degree of the protein response to estrogen was comparatively blunted in the CC-incorporated cycles. Endometrial thickness and pregnancy outcomes correlated positively with the expression of the protein. The 64.0-kDa hUF protein perhaps plays a role in endometrial receptivity to support pregnancy. Failure of pregnancy despite documented ovulation in CC-stimulated cycles may be due to its attenuating effects on expression of the protein. (Fertil Steril� 2007;87:343–50. ©2007 by American Society for Reproductive Medicine.

Assessment of oocyte quality in polycystic ovarian syndrome and endometriosis by spindle imaging and reactive oxygen species levels in follicular fluid and its relationship with IVF-ET outcome

Objectives: The aim of this study is to examine meiotic spindle in oocytes along with reactive oxygen species (ROS) levels in follicular fluid of women undergoing IVF and to correlate these findings with embryo quality and pregnancy outcome. Materials and Methods: 167 women aged 25-35 years with endometriosis (Group A), polycystic ovarian syndrome (PCOS) (Group B) and tubal block (Group C) were included. Long protocol downregulation using recombinant follicular stimulating hormone was used for ovarian stimulation. Aspirated follicular fluid containing mature oocytes were analyzed for ROS levels and the oocytes were assessed for the presence of meiotic spindle using Cri-OosightTM Polscope. Fertilization, embryo quality, endometrial assessment, and final pregnancy outcome were assessed. Results: Meiotic spindles were visualized in a higher proportion of mature oocytes retrieved from women with endometriosis (66%) as compared to those with PCOS (50.5%) and tubal block (62.3%). ROS levels were also observed to be significantly less in the follicular fluid of oocytes in women with endometriosis (Group A) as compared to the other two groups (P ≤ 0.001). However, pregnancy rates were observed to be lower in Group A (32%) than Groups B (39%) and C (44%), respectively. Within each group, oocytes with spindle visualization yielded a higher number of Grade 1 embryos (P < 0.05) as well as lower ROS levels in follicular fluid (P ≤ 0.001) as compared to those where spindle could not be visualized. Conclusions: There was good correlation between spindle imaging and ROS levels as reliable predictors of oocyte assessment. Women with endometriosis had low ROS levels and good spindle imaging results suggesting a possible role of endometrial receptivity accounting for lower pregnancy rates in these women. Poor oocyte quality, as reflected by higher mean ROS levels and low number of oocytes with spindle visualization, could be the factor impeding pregnancy in women with PCOS as compared to women with tubal block

Mechanism of antiulcer effect of neem (Azadirachta indica) leaf extract: effect on H<SUP>+</SUP>-K<SUP>+</SUP>-ATPase, oxidative damage and apoptosis

The mechanism of the antiulcer effect of Neem leaf aqueous extract to block gastric lesions in rat has been studied with emphasis on acid secretion, oxidative damage and apoptosis. The extract dose-dependently inhibits gastric lesions induced by restraint-cold stress, indomethacin and ethanol. In stress ulcer model, it is more effective than ranitidine but less effective than omeprazole. It also dose-dependently blocks pylorus ligation and mercaptomethylimidazole-induced acid secretion. In the pylorus-ligation model, it is less effective than omeprazole but as effective as ranitidine. It inhibits H+-K+-ATPase activity in vitro in concentration-dependent manner to inhibit acid secretion. Oxidative membrane damage by hydroxyl radical (&#8727;OH) as measured by lipid peroxidation in stress ulcer is significantly blocked by leaf extract. Stress-induced apoptotic DNA fragmentation is also protected. The extract also prevents OH-mediated mucosal DNA damage in vitro by scavenging the OH. Neem leaf extract, thus, offers antiulcer activity by blocking acid secretion through inhibition of H+-K+-ATPase and by preventing oxidative damage and apoptosis

Gastroprotective effect of Neem (Azadirachta indica) bark extract: possible involvement of H<SUP>+</SUP>-K<SUP>+</SUP>-ATPase inhibition and scavenging of hydroxyl radical

The antisecretory and antiulcer effects of aqueous extract of Neem (Azadirachta indica) bark have been studied along with its mechanism of action, standardisation and safety evaluation. The extract can dose dependently inhibit pylorus-ligation and drug (mercaptomethylimidazole)-induced acid secretion with ED<SUB>50</SUB> value of 2.7 and 2 mg Kg<SUP>-1</SUP> b.w. respectively. It is highly potent in dose-dependently blocking gastric ulcer induced by restraint-cold stress and indomethacin with ED<SUB>50</SUB> value of 1.5 and 1.25 mg Kg<SUP>-1</SUP> b.w. respectively. When compared, bark extract is equipotent to ranitidine but more potent than omeprazole in inhibiting pylorus-ligation induced acid secretion. In a stress ulcer model, it is more effective than ranitidine but almost equipotent to omeprazole. Bark extract inhibits H<SUP>+</SUP>-K<SUP>+</SUP>-ATPase activity in vitro in a concentration dependent manner similar to omeprazole. It offers gastroprotection against stress ulcer by significantly preventing adhered mucus and endogenous glutathione depletion. It prevents oxidative damage of the gastric mucosa by significantly blocking lipid peroxidation and by scavenging the endogenous hydroxyl radical (&#8730;OH)-the major causative factor for ulcer. The &#8730;OH-mediated oxidative damage of human gastric mucosal DNA is also protected by the extract in vitro. Bark extract is more effective than melatonin, vitamin E, desferrioxamine and &#945;-phenyl N-tert butylnitrone, the known antioxidants having antiulcer effect. Standardisation of the bioactive extract by high pressure liquid chromatography indicates that peak 1 of the chromatogram coincides with the major bioactive compound, a phenolic glycoside, isolated from the extract. The pharmacological effects of the bark extract are attributed to a phenolic glycoside which is apparently homogeneous by HPLC and which represents 10% of the raw bark extract. A single dose of 1g of raw extract per kg b.w. (mice) given in one day and application of 0.6g raw extract per kg b.w. per day by oral route over 15 days to a cumulative dose of 9g per kg was well tolerated and was below the LD<SUB>50</SUB>. It is also well tolerated by rats with no significant adverse effect. It is concluded that Neem bark extract has therapeutic potential for the control of gastric hyperacidity and ulcer