Generation of mice with a conditional null allele for Wntless

The Wnt‐signaling pathway is necessary in a variety of developmental processes and has been implicated in numerous pathologies. Wntless (Wls) binds to Wnt proteins and facilitates Wnt sorting and secretion. Conventional deletion of Wls results in early fetal lethality due to defects in body axis establishment. To gain insight into the function of Wls in later stages of development, we have generated a conditional null allele. Homozygous germline deletion of Wls confirmed prenatal lethality and failure of embryonic axis formation. Deletion of Wls using Wnt1‐cre phenocopied Wnt1 null abnormalities in the midbrain and hindbrain. In addition, conditional deletion of Wls in pancreatic precursor cells resulted in pancreatic hypoplasia similar to that previously observed after conditional β‐catenin deletion. This Wls conditional null allele will be valuable in detecting novel Wnt functions in development and disease. genesis 48:554–558, 2010. © 2010 Wiley‐Liss, Inc.     

Procathepsin D expression correlates with invasive and metastatic phenotype of MDA-MB-231 derived cell lines

Procathepsin D (pCD) is a glycoprotein secreted abundantly by cancerous cells with a documented role in tumor development. The levels of pCD in primary tumors are highly correlated with an increased incidence of metastasis. Our earlier studies have shown that pCD exerts its effect on cancer cells through its activation peptide (AP) and involves both autocrine and paracrine modes of action. In this study, we analyzed the expression and role of pCD in MDA-MB-231 and its derived cell lines 1833 and 4175 possessing discrete metastatic abilities. Our results demonstrated a direct relationship between expression and secretion of pCD to the differential invasive potential of these cells. Also, the cell lines responded to AP treatment by enhancing their invasive potential, proliferation and induction of secretion of various cytokines, suggesting that pCD plays a role in metastasis through its AP region.     

Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.     

The role of a heat shock protein from V. cholerae 0139 in the gut immune response

An immunodominant heat shock protein (Hsp 24) was purified from Vibrio cholerae O139 at 42 degrees C and used as an immunomodulator for studying the gut immune response. T cell clone and T cell line specific for the Hsp 24 were generated from the lymphocytes of lamina propria and intra-epithelial lymphocytes of mice orally infected with V. cholerae O139, respectively. The T cell clone was TCR alphabeta(+), CD4(+) and appeared to play an important role in the functioning of gut B-lymphocytes. The T cell line had heterogenous population of CD8+ and CD4+ cells, most of which were found to be TCR alphabeta(+) and a minor population was TCR gammadelta(+). The lymphokine profile of T cell line showed IFN-gamma to be the most abundant lymphokine followed by IL-2 and IL-4. The possible involvement of alternative pathway of activation for T cell clone was also addressed in this study. The splenocytes showed an up-regulation of their CD2 receptor expression on stimulation with the Hsp-24. The pattern of lymphokines released by splenocytes stimulated with the Hsp-24 showed no particular cell type to be responsible for mounting immune response. Thus, there is involvement of both, mucosal and peripheral arm of the immune system.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

Structural and functional studies on Ribonuclease S, retro S and retro-inverso S peptides

Ribonuclease S peptide and S protein offer a unique complementation system to understand the finer features of molecular recognition. In the present study the S peptide (1-16), and its retro and retro-inverso analogs have been analyzed for their structural and biological attributes. RPHPLC, CD, and NMR analyses have revealed that the physicochemical and conformational properties of the S peptide are distinct from those of its retro and retro-inverso analogs. On the functional side, while the S peptide complemented the S protein to give RNase activity, was recognized by anti-S peptide antibodies and induced T cell proliferation, neither the retro nor the retro-inverso S peptides could do so.     

Identification and association of ISSR markers for thermal stress in polyvoltine silkworm Bombyx mori

Evaluation of genetic resources is an essential prerequisite for their effective utilization. In India, the tropical climate prevails in most of the sericultural belts, where temperature goes beyond the ambient during summer, adversely affecting the silkworm rearing. Although polyvoltine silkworms are poor in silk content, they are mostly tolerant to tropical conditions and diseases. With an aim to identify potential silkworm races/breeds specific to thermo-tolerance for their effective utilization in breeding programme, 15 selected polyvoltine silkworm races were studied for their thermo-tolerance behaviour. Their genomic DNA samples were analyzed for ISSR-PCR using 15 selected primers. The UPGMA analysis based on Nei and Li algorithm has clustered the 15 silkworm races into five groups and one isolate. ALSCAL-multidimensional scaling has not only supported the information generated by the dendrogram, but it has made the genetic distance among races more clear and substantiating their status in terms of thermal stress where pupation rate was taken as indicator. Further, discriminant function analysis (DFA) was done with three groups of silkworms classified for thermal stress viz. susceptible, moderately tolerant and tolerant. The canonical correlation value was estimated to be 0.987 (Wilk's lambda = 0.004; chi2 = 36.044, p < 0.05). DFA clearly discriminated the above three groups. Beta statistics with t value and its significance for the markers identified through stepwise multiple regression analysis (MRA) revealed a total of five bands (807(1300), 808(3000), 808(4000), 834(4000), and 834(3000)) showing correlation with pupation rate after thermal treatment. Out of them, marker 8083000 showed maximum and highly significant correlation (r = 0.757, p < 0.001, t = 4.182) with pupation rate among the silkworm races. The identified putative markers are being used to develop DNA marker to be used in evolving thermo-tolerant silkworm breeds using marker assisted selection programme.     

Galactose-Specific Fimbrial Adhesin of Enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis>: A Possible Aggregative Factor

A galactose-specific adhesin was isolated from the fimbriae of an enteroaggregative Escherichia coli (EAEC) strain. The adhesin was found to be a high molecular weight aggregate of the 18-kDa monomer. The dimeric (36 kDa) and tetrameric (76 kDa) forms appeared in sodium dodecyl sulphate polyacrylamide gel electrophoresis when a higher concentration of the adhesin was used. The IgG_AD (IgG against adhesin) obtained from the immune sera raised in rabbits against purified adhesin could detect all three forms of the adhesin even from the crude fimbrial preparation. The IgG_AD failed to recognize the adhesin in the presence of galactose, thereby suggesting the antibody-binding site and the sugar-binding site on the adhesin might be same or overlapping. Furthermore, the IgG_AD could localize the adhesin exclusively on the fimbriae as observed in immunogold electron microscopy. The aggregative adherence of the bacteria to HEp-2 cells was reduced to 70% in the presence of the IgG_AD. A glycoprotein (34 kDa) present in the membrane fraction of HEp-2 cells interacted with the purified adhesin in a galactose-specific manner. The IgG_AD could recognize the adhesin from the crude fimbrial preparation of 9 out of 10 clinical isolates of EAEC strains but failed to identify any protein from the crude fimbrial preparation of Salmonella typhimurium (fim +ve as well as fim −ve strain), Vibrio cholerae (WO7) or Escherichia coli DH5α.