PTP-PEST couples membrane protrusion and tail retraction via VAV2 and p190RhoGAP

Cell motility is regulated by a balance between forward protrusion and tail retraction. These phenomena are controlled by a spatial asymmetry in signals at the front and the back of the cell. We show here that the protein-tyrosine phosphatase, PTP-PEST, is required for the coupling of protrusion and retraction during cell migration. PTP-PEST null fibroblasts, which are blocked in migration, exhibit exaggerated protrusions at the leading edge and long, unretracted tails in the rear. This altered morphology is accompanied by changes in the activity of Rho GTPases, Rac1 and RhoA, which mediate protrusion and retraction, respectively. PTP-PEST null cells exhibit enhanced Rac1 activity and decreased RhoA activity. We further show that PTP-PEST directly targets the upstream regulators of Rac1 and RhoA, VAV2 and p190RhoGAP. Moreover, we demonstrate that the activities of VAV2 and p190RhoGAP are regulated by PTP-PEST. Finally, we present evidence indicating the VAV2 can be regulated by integrin-mediated adhesion. These data suggest that PTP-PEST couples protrusion and retraction by acting on VAV2 and p190RhoGAP to reciprocally modulate the activity of Rac1 and RhoA.     

Molecular characterization of beta-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

The beta-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reaction-restriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A beta-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a 'B-like' enzyme. This is the first study to have investigated the genetic heterogeneity of the beta-lactamase genes of Y. enterocolitica.     

Antioxidant response in sesame plants grown on industrially contaminated soil: effect on oil yield and tolerance to lipid peroxidation

The plants of sesame white (Sesamum indicum L. var. T55) grown on tannery sludge (TS) contaminated soil have shown that Cr level in the seeds was found below detection limits in 10% and 25% TS, however, the levels of Ni, Pb and Cd were found above the recommended limits. In roots, the level of antioxidants increased in the plants grown upto 35% TS at 30d over their respective controls. Total chlorophyll content increased significantly (p<0.5) in the plants (leaves) grown on lower sludge amendments (upto 35% TS at 30d and 25% TS at 60d) over their respective controls. In addition, the oil content increased (35% increase over control) in the plants grown on 35% TS. No significant change was observed in thiobarbituric acid reactive substances (TBARS), a lipid peroxidation index, in the plants (upto 50% TS). The number of trichomes in the leaves of treated plants was found more than control. In lower and upper leaves surfaces, the anterior end of the trichomes was found acute tipped and bent downwards, whereas, the trichome tip was straight and blunt in control. The stomata on upper and lower surfaces of the leaves were found partially or totally closed in the plants grown on 100% TS as compared to control. The toxicity was observed at higher amendments which are evident from the observed morphological changes and decrease in chlorophyll content. This study concludes that it is not advisable to grow the plants on contaminated area, besides its healthy growth.     

Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella. S. K. Singh and M. K. Rai contributed equally to this work.     

Sex‐specific effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic/steroid‐metabolizing enzymes and antioxidant status

Liver has evolved complex enzymatic mechanisms to detoxify a wide array of xenobiotic substances, ranging from dietary components to environmental toxins to pharmaceuticals. Activities of many steroid‐metabolizing enzymes in adult rat liver microsomes are sexually differentiated. Toxic effects of lead and cadmium on hepatic tissue have been well established in our earlier studies. We thus monitored the effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic‐ and steroid‐metabolizing enzyme activities in both male and female F1 generation postnatal day (PND) 56 rats. Adult pregnant female rats were treated subcutaneously [0.05 mg/(kg body wt. day)] with sodium acetate (control group), lead acetate, and cadmium acetate separately and in combination throughout the gestational and lactational period. Hepatic phase I xenobiotic‐metabolizing enzymes (NADPH‐ and NADH‐cytochrome c reductase) activities significantly decreased significantly in all the metal‐treated groups in both PND 56 male and female rats as compared with the control group. Hepatic phase II enzymes (uridine diphosphate‐glucuronosyl transferase, γ‐glutamyl transpeptidase, glutathione‐S‐transferase, 17‐β‐hydroxysteroid oxidoreductase) were also highly susceptible to all the metal‐treated groups. The observed alterations in the oxidative stress and biochemical parameters in the liver of F1 generation male and female rats resulted from an independent effect of lead and/or cadmium and also from their interaction. Results suggest that early developmental exposure to lead and cadmium both alone and in combination can suppress the hepatic xenobiotic‐metabolizing enzyme activities in the liver of F1 generation male and female rats in a sex‐dependent manner. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:419–431, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20305     

Growth,metal accumulation and yield performance of Brassica campestris L. (cv. Pusa Jaikisan) grown on soil amended with tannery sludge/fly ash mixture

Application of sewage sludge and fly ash (FA) to soil is gaining practice and has become an alternative to chemical fertilizers in many countries. The present study aimed to assess the potential of fly ash–tannery sludge (TS) mixture in two ratios {4 (TS):1 (FA), denoted as A and 4 (TS):2 (FA) denoted as B} and different amendments were made with soil to study the yield and metal availability and their uptake by Brassica campestris L. (cv. Pusa Jaikisan). Experimental data showed that increase in dry matter yield and oil content of B. campestris was noted in lower amendments of soil with mixture A. However, total chlorophyll content of the leaf increased significantly, whereas carotenoid content showed non-significant increase in all the amendments with mixtures A and B as compared to control. The translocation of most of the tested metals (Pb, Mn, Cd, Ni, and Fe) in the shoot of the plant was found higher except Cr, Cu, and Co. Overall, experimental results showed that mixture A (≥10%) was found to be suitable due to better yield and less accumulation of metal in the seed of B. campestris.     

Acidophilic tannase from marine Aspergillus awamori BTMFW032

Aspergillus awamori BTMFW032, isolated from sea water, produced tannase as extracellular enzyme under submerged culture conditions. Enzyme with a specific activity of 2761.89 IU/mg protein, a final yield of 0.51 %, and a purification fold of 6.32 was obtained after purification to homogeneity by ultrafiltration and gel filtration. SDS-PAGE analyses under non- reducing and reducing conditions yielded a single band of 230 kDa and 37.8 kDa, respectively, indicating presence of six identical monomers. pI of 4.4 and 8.02 % carbohydrate content in the enzyme were observed. Optimal temperature was 30oC, although the enzyme was active at 5-80 oC. Two pH optima, pH 2 and pH 8, were recorded and the enzyme was stable only at pH 2.0 for 24 h. Methylgallate recorded maximal affinity and K(m) and V(max) were recorded, respectively, as 1.9 X 10?3 M and 830 micronmol/min. Impact of several metal salts, solvents, surfactants, and typical enzyme inhibitors on tannase activity were determined to establish the novelty of the enzyme. Gene encoding tannase isolated from A. awamori is 1.232 kb and nucleic acid sequence analysis revealed an open reading frame consisting of 1122 bp (374 amino acids) of one stretch in -1 strand. In-silico analyses of gene sequences and comparison with reported sequences of other species of Aspergillus indicated that the acidophilic tannase from marine A. awamori is differs from that of other reported species.     

Identification of Novel Synthetic Toll-like Receptor 2 Agonists by High Throughput Screening

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit.     

Biochemical and molecular effects of gestational and lactational coexposure to lead and cadmium on ovarian steroidogenesis are associated with oxidative stress in F1 generation rats

Few studies have characterized the molecular and biochemical mechanisms involved in ovarian steroidogenesis disruption by heavy metals, such as lead and cadmium coexposure, on F1 generation offspring. In this study, adult pregnant female rats were treated subcutaneously (0.05 mg/kg of body weight per day) with sodium acetate (control), lead acetate, and cadmium acetate separately and in combination throughout gestational and lactational period, and all animals from each of the experimental groups were sacrificed by decapitation on postnatal day 56 for various assays. The activities of key steroidogenic enzymes (17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase) decreased in all the metal-treated groups. But the most significant decrease in the activities was observed in the cadmium-treated group, whereas the combined exposure group showed an intermediate effect. Serum estradiol and progesterone levels were also significantly altered in all the metal-treated groups, with the cadmium-exposed group showing maximum reductions as compared with the control group. The inhibitory effects of lead and cadmium on ovarian steroidogenic acute regulatory protein (StAR) mRNA levels along with CYP11 mRNA levels were also observed. Ovarian cholesterol content measured also showed significant depletion in all the metal-treated groups, with the cadmium-exposed group showing the maximum depletion. The activities of ovarian enzymatic antioxidants, such as superoxide dismutase, catalase, and glutathione peroxidase, were all significantly diminished along with significant depletion in nonenzymatic antioxidants. Lipid peroxidation was elevated significantly in all the metal-treated groups. In conclusion, lead and cadmium inhibit ovarian steroidogenesis by downregulating StAR gene expression along with inhibiting activities of steroidogenic enzymes and antioxidant system.     

Inheritance of resistance and cross resistance pattern in indoxacarb-resistant diamondback moth Plutella xylostella L.

Leaf-dip assay of Plutella xylostella against indoxacarb showed that the concentration that produced 50% mortality (LC₅₀) of indoxacarb ranged from 20.1 to 11.9 ppm, with highest in Nasik and lowest levels in Coimbatore strains. In selection studies, the LC₅₀ of indoxacarb was 18.5 ppm at generation 1 (G1), which increased to 31.3-fold (167.8 ppm) resistance after ten exposed generations (G10) as compared to unexposed. The LC₅₀ of quinalphos was 74.4 ppm, which increased to 10.0-fold (631.5 ppm) resistance after G10. The LC₅₀ of cypermethrin resistant strain resulted in an 11.5-fold increase in resistance after G10. In P. xylostella , heritability (h²) after ten generations of selection was estimated at 0.4. The number of generations required for tenfold increase in LC₅₀ (1/R) were 6.7. The response to indoxacarb selection in P. xylostella was 0.2 and the selection differential was estimated as 0.4. The phenotypic standard deviation was 0.2. Reciprocal crosses between indoxacarb-resistant and susceptible strains showed that the inheritance of indoxacarb resistance was autosomal. The degree of heritability (D_LC) (0.4, 0.4) indicated incomplete recessive inheritance of indoxacarb resistance.