Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

Role of chemokines in Leishmania infection

Chemokines are a growing group of chemoattractant cytokines that play important roles in physiological as well as pathological processes. Their roles in various aspects of pathogenesis and inflammation have come to light in the past decade or so. It is becoming increasingly clear that chemokines play a major, perhaps decisive role in Leishmania infections. In this review, we recapitulate important works linking the chemokine system with relation to visceral and cutaneous leishmaniasis over the past decade and attemptto put it all together to propose a single yet unfinished model to account for all the findings.     

Anticoagulant and antiplatelet agents: their clinical and device application(s) together with usages to engineer surfaces

An essential aspect of the treatment of patients with cardiovascular disease is the use of anticoagulant and antiplatelet agents for the prevention of further ischaemic events and vascular death resulting from thrombosis. Aspirin and heparin have been the standard therapy for the management of such conditions to date. Recently, numerous more potent platelet inhibitors together with anticoagulant agents have been developed and tested in randomized clinical trials. This article reviews the current state of the art of antiplatelet and anticoagulant therapy in light of its potential clinical efficacy. It then focuses on the usages of these agents in order to improve the performance of clinical devices such as balloon catheters, coronary stents, and femoropopliteal bypass grafting and extra corporeal circuits for cardiopulmonary bypass. The article then goes on to look at the usage of these agents more specifically heparin, heparan, hirudin, and coumarin in the development of more biocompatible scaffolds for tissue engineering.     

Synthesis and antihyperglycemic activity profiles of novel thiazolidinedione derivatives

A number of thiazolidine-2,4-diones derivatives having carboxylic ester appendage at N-3 were synthesized and their antihyperglycemic activity was evaluated. Many of these derivatives as well as their corresponding carboxylic acid showed significant improvement on post-prandial hyperglycemia in normal rats, in contrast to their poor agonist activity at PPARgamma.     

GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.     

Associative diazotrophs of pearl millet (Pennisetum glaucum) from semi arid region--isolation and characterization

Diversity of the native diazotrophs associated with the rhizosphere of pearl millet (P. glaucumn), grown in nutritionally poor soils of semi-arid regions was studied with a view to isolate effective nitrogen fixing and plant growth stimulating bacteria with root associative characteristics. The native population varied from 10(3)-10(4) g(-1) of rhizosphere soil after 40 d growth and belonged to genera Azospirillum, Azotobacter and Klebsiella. Another non-diazotrophic root associative group was Pseudomonas sp., which also produced IAA and enhanced plant growth. Some of these rhizobacteria showed high in vitro acetylene reduction activity along with production of indole acetic acid. Out of 11 selected diazotrophs used as seed inoculants, M10B (Azospirillum sp.), M11E (Azotobacter sp.) and M12D4 (Klebsiella sp.) resulted in significant increase in total root and shoot nitrogen at 45 and 60 days of plant growth under pot culture conditions.     

Endothelial barrier strengthening by activation of focal adhesion kinase

Endothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening.     

A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical

The mechanism of the antiulcer effect of omeprazole was studied placing emphasis on its role to block oxidative damage and apoptosis during ulceration. Dose-response studies on gastroprotection in stress and indomethacin-induced ulcer and inhibition of pylorus ligation-induced acid secretion indicate that omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent mechanism for its antiulcer effect. Time course studies on gastroprotection and acid reduction also indicate that omeprazole almost completely blocks lesions at 1 h when acid inhibition is partial. The severity of lesions correlates well with the increased level of endogenous hydroxyl radical (*OH), which when scavenged by dimethyl sulfoxide causes around 90% reduction of the lesions, indicating that *OH plays a major role in gastric damage. Omeprazole blocks stress-induced increased generation of *OH and associated lipid peroxidation and protein oxidation, indicating that its antioxidant role plays a major part in preventing oxidative damage. Omeprazole also prevents stress-induced DNA fragmentation, suggesting its antiapoptotic role to block cell death during ulceration. The oxidative damage of DNA by *OH generated in vitro is also protected by omeprazole or its analogue, lansoprazole. Lansoprazole when incubated in a *OH-generating system scavenges *OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak at m/z 385, which is 16 mass units higher than that of lansoprazole (m/z 369). The product shows no additional aromatic proton signal for aromatic hydroxylation in (1)H NMR. The product absorbing at 278 nm shows no alkaline shift for phenols, thereby excluding the formation of hydroxylansoprazole. The product is assigned to lansoprazole sulfone formed by the addition of one oxygen atom at the sulfur center following attack by the *OH. Thus, omeprazole plays a significant role in gastroprotection by acting as a potent antioxidant and antiapoptotic molecule.