Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC₅₀ value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC₅₀ value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10⁻⁷ M⁻¹ s⁻¹ and 4.0 × 10⁻⁹ M⁻¹ s⁻¹ for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin–apigenin binding was 1:1 and binding the binding constant (K_d) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G₂/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α–β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.     

Prolactin secreting cells in the hypophysis of the brown spiny mouseMus platythrix (Bennett)

Prolactin secreting cells are identified in thepars distalis of Mus platythrix by conventional methods of light and electron microscopy. Two types of prolactin secreting cells are recognised. These types are estrone-sensitive, mammotrophic type I, and luteotrophic type II, respectively. Histochemical analysis revealed that the cells are rich in RNA, basic proteins, alkaline phosphatase and are resistant to extraction with 0·5% trichloroacetic acid. Quantitative data showed that the prolactin secreting cells increase during pregnancy, lactation and estrone treatment. Estrone at low dose levels caused immense hyperplasia whereas at higher levels there was no corresponding increase in the percentage of type I cells. Ultrastructurally, prolactin secreting cells are characterised by the presence of stacked endoplasmic reticulum, oval or irregular secretory granules. The Golgi apparatus is seen rich in vacuolar system.     

Physical mapping of two Xp markers DXS16 and DXS143

Summary Lymphocyte karyotyping of an infant girl with the clinical features of microphthalmia, iridoschisis, goiter, hip joint dysplasia, labium synechia and craniotabes revealed an Xp deletion. The lymphocyte karyotypes of the parents were normal. Bromodeoxyuridine incorporation studies showed that, in 42 out of 43 metaphases, the deleted X chromosome was late replicating. In one metaphase, the normal X chromosome was observed to be allocyclic. Using DNA markers from the Xp22 region, the breakpoint was assigned distal to DXS16 (pXUT23) and proximal to DXS143 (dic56). Dosage intensity measurements confirmed that the STS gene and the DNA marker DXS31 were involved in the deleted area. Restriction fragment length polymorphism analysis revealed that the paternally derived X-chromosome was deleted.     

A simple quantitative method for the estimation of free ecto-sulfhydryl groups of spermatozoa

A simple rapid quantitative method has been developed for the estimation of sperm ecto-SH groups on the basis of their high affinity binding to the mercurial: [203Hg]p-chloromercuriphenylsulfonic acid (PCMPS) used as a surface probe. The thiol reagent did not penetrate the sperm plasma membrane, as evidenced by the extremely rapid time course of the binding reaction and undetectable uptake of [203Hg]PCMPS by intact goat spermatozoa. The binding reaction was not due to contaminating broken or damaged cells, if any. The method consists of incubating of highly motile goat spermatozoa with PCMPS in a modified Ringer solution at 37 degrees C for 5 min, agglutination of the labelled cells with polyethyleneimine (100 micrograms/ml) and filtration and washing of the cell suspension through Whatman No. 1 filter discs under mild vacuum. The binding interaction is proportional to cell concentration, specific and saturable at 50 microM PCMPS. The method is capable of estimating free ecto-SH as low as 25 pmoles. Spermatozoa possess 286 +/- 61 pmoles of free ecto-SH groups/10(6) cells. Scatchard analysis showed the presence in goat spermatozoa of multiple classes of ecto-SH groups differing in their affinity for PCMPS.     

Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Antihyperglycemic effect of iridoid glucoside, isolated from the leaves of Vitex negundo in streptozotocin-induced diabetic rats with special reference to glycoprotein components

The aim of present study was to isolate an iridoid glucoside from the leaves of Vitex negundo and evaluates its effects on dearrangement in plasma and tissues glycoprotein components in streptozotocin-induced diabetic rats. The levels of blood glucose, plasma and tissues glycoproteins such as hexose, hexosamine, fucose and sialic acid were significantly increased whereas plasma insulin levels were significantly decreased in diabetic rats. On oral administration of iridoid glucoside at a concentration of 50 mg/kg b.w. once daily to diabetic rats for the period of 30 days, reversed the above-mentioned hyperglycemia-induced biochemical changes to near normal levels. The anti-hyperglycemic effect of iridoid glucoside was comparable with glibenclamide, a known hypoglycemic drug. Based on the results obtained from the present study, it may be concluded that iridoid glucoside possesses significant productive effect on glycoprotein metabolism in addition to its antidiabetic effect.     

Leptin downregulates ethanol-induced secretion of proinflammatory cytokines and growth factor

Inflammatory mediators, including cytokines and growth factors are associated with the pathology of chronic liver diseases. The aim of our present work was to study the effect of exogenous leptin and/or ethanol on the secretion of TNF-alpha, IL-6 and TGF-beta1 both in vivo and in vitro. Forty eight hours after the exposure to ethanol (500 mM) significantly elevated the secretion of TNF-alpha, IL-6 and TGF-beta1 in the cell-free culture supernatant (HepG2 and mouse HCC cell lines), which were decreased on leptin (31.2 nM) treatment. Similarly, leptin administration to ethanol (6.32 g kg(-1) body weight) fed mice for 45 days significantly lowered the concentration of these cytokines in the circulation; however, leptin alone (230 microg kg(-1) body weight i.p. administered every alternate day for the last 15 days) had no such significant effect on cytokine secretion both in vivo and in vitro conditions. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system in alcoholic liver disease.     

Arbuscular Mycorrhizal Fungi Community Structure,Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity.