Search PubMed⌕ Search

Biomedical subjects

A Chawla

Publications and source records attributed to A Chawla.

At least 55 records · Page 3Linked to original sources
PubMed1997

Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues.

The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed syntaxin. These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery. Synaptobrevin/VAMP and syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells. However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues. Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple syntaxins and synaptobrevins/VAMPs in vitro. SNAP-23 is 59% identical with SNAP-25. Unlike SNAP-25, SNAP-23 was expressed in all tissues examined. These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.

Amino Acid Sequence↗
PubMed1996

Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma.

Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARgamma and C/EBPalpha. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARalpha and RARgamma; during adipocyte differentiation, RARalpha gene expression was nearly constant, whereas RARgamma1 mRNA and protein levels dramatically decreased. Ectopic expression of RARgamma1 extended the period of effectiveness of RA by 24 to 48h; RARalpha expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARgamma1 and PPARgamma2 proteins had already been expressed and resulted in the loss of PPARgamma proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARgamma. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.

3T3 Cells↗
PubMed1995

Effect of olsalazine on sodium-dependent bile acid transport in rat ileum.

Olsalazine (OLZ), a relatively new form of 5-aminosalicylic acid (5-ASA), is being used for the treatment of colitis. A major side effect of olsalazine is diarrhea, reported in 12-25% of patients. One suggested mechanism for this side effect is enhanced ileal water and electrolyte secretion. We propose that OLZ may also inhibit ileal bile acid (BA) transport, resulting in choleretic diarrhea. This would result in excess BAs reaching the colon, with consequent BA-induced secretory diarrhea. Therefore, we studied the effect of OLZ on rat ileal absorption of taurocholate. BA uptake was determined in rat ileal segments, everted sacs, brush border membrane vesicles (BBMV), and Xenopus laevis oocytes. Segments and everted sacs were treated with 5 mM OLZ for 30 min prior to and throughout 10-min taurocholate (Tc) uptake. Terminal ileal BBMV were used to study the effect of OLZ on sodium-dependent bile acid uptake independent of cellular metabolism. Direct effects on the bile acid carrier were examined using Xenopus laevis oocytes expressing the cloned apical rat ileal BA transporter. In ileal segments 5 mM OLZ inhibited 10-min Tc uptake by 69.4 +/- 8.8% (P < 0.01) (N = 10 animals). Increasing concentrations of OLZ resulted in a dose-dependent inhibition of Tc uptake. Ten-minute Tc uptake with 0.5, 1.0, 2.0, 2.5, and 5 mM OLZ was inhibited by 13.5, 39.6, 49.7, and 70.5%, respectively. In BBMV, OLZ inhibited 45-sec Tc uptake in a dose-dependent manner but did not effect Na-dependent L-alanine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminosalicylic Acids↗
PubMed1995

Effect of N-3 polyunsaturated fatty acid supplemented diet on neutrophil-mediated ileal permeability and neutrophil function in the rat.

RATIONALE AND OBJECTIVE: Fish oil, rich in n-3 polyunsaturated fatty acids, can alter leukotriene production and hence neutrophil function, factors which may be important in the inflammation of Crohn's disease (CD). Therefore we studied the effect of dietary PUFA on neutrophil mediated ileal inflammation and neutrophil function in the rat. METHODS: Animals were ad libitum-fed pellet diets containing 9.5% fish oil (menhaden oil, rich in n-3 PUFA) with 0.5% safflower oil, 10% safflower oil (rich in n-6 PUFA) or standard chow (6% fat) for 50 days prior to the study. Weight and circulating leukocyte and total neutrophil counts were identical in all three groups. Neutrophil mediated ileal inflammation induced by formyl-methionyl-leucyl-phenylalanine (fMLP) perfusion was evaluated by measuring macromolecular uptake of radiolabelled dextran (MW 70,000) and changes in mucosal neutrophil infiltration. RESULTS: The fish oil diet group showed no difference in fMLP-induced permeability changes relative to the Chow Control group. However, the Safflower Oil supplemented diet group had a reduced permeability response (p < 0.01). Mirroring the permeability changes, there was diminished mucosal neutrophil infiltration in the Safflower Oil group following ileal perfusion with fMLP (< .005). Chemotaxis and chemiluminescence, two important neutrophil functions, were also significantly suppressed in the Safflower Oil animals (p < 0.05). CONCLUSIONS: The failure of a n-3 PUFA enriched diet to diminish the ileal inflammatory response to a bacterial peptide and suppress neutrophil function in the rat suggests such therapy would not be expected to be highly successful in CD. However, it requires confirmation in man, especially under the more complicated inflammatory conditions found in CD. On the other hand, the decreased neutrophil mediated responses with a high linoleic acid (n-6 PUFA) diet warrant further investigation.

Animals↗
PubMed1995

The frequency and influence of gallbladder varices on gallbladder functions in patients with portal hypertension.

Gallbladder varices have been reported in patients with portal hypertension. The exact frequency and significance of these collaterals in patients with cirrhotic and noncirrhotic portal hypertension is not known. One hundred and two patients with portal hypertension [38 with cirrhosis, 29 with noncirrhotic portal fibrosis (NCPF) and 35 with extrahepatic portal vein obstruction (EHPVO)] and 25 healthy controls were studied. Gallbladder varices were seen at ultrasound as tortuous, dilated vessels in the wall or in the bed of the gallbladder. In 35 patients (19 patients with and 16 without gallbladder varices) and in 10 healthy controls, gallbladder functions were studied by determining fasting volume (FV) and then residual volume (RV) every 10 min over 1 h after giving a liquid meal of 420 k.cal. Ejection fraction (EF) was computed as a percentage by the formula: FV--RV/FV x 100. Twenty four (24%) patients had gallbladder varices: Five (13%) with cirrhosis, seven (24%) with NCPF, and 12 (34%) with EHPVO. FV in EHPVO patients was seen significantly more than in cirrhotics (31.6 +/- 15.4 vs 19.3 +/- 6.0 ml, p < 0.05). The RV and EF were not different in the three groups of patients compared with the controls. The EF was similar in patients with or without gallbladder varices (63.3 +/- 10.2% vs 64.6 +/- 10.4%). Gallbladder varices are often seen in portal hypertension, more often in EHPVO patients, and these collaterals cause some gallbladder stasis but do not impede gallbladder function and hence seem unlikely to contribute to gallstone formation.

Adolescent↗
PubMed1994

Peroxisome proliferator and retinoid signaling pathways co-regulate preadipocyte phenotype and survival.

Culture of mouse 3T3-L1 preadipocytes in medium containing delipidated bovine calf serum caused the cells to elongate and divide after reaching confluency. The continued proliferation correlated with sustained expression of the c-myc gene, which was repressed in control cells. Exposure of the cells to activators of peroxisome proliferator-activated receptor (PPAR), including clofibrate, WY-14,643, and 5,8,11,14-eicosatetraynoic acid, reversed and prevented the effects of culturing preadipocytes in delipidated serum. Continued exposure to PPAR activators led to adipose conversion, during which PPAR and its heterodimerization partner (retinoid X receptor) were induced. Retinoic acid (RA) had no effect on the growth or survival of preadipocytes grown in the presence of normal bovine serum. However, treatment of cells cultured in delipidated serum with RA caused death of the cells by apoptosis. Thus, preadipocyte phenotype and survival are regulated by activators of nuclear hormone receptors.

3T3 Cells↗
PubMed1994

Retinoic acid prevents downregulation of ras recision gene/lysyl oxidase early in adipocyte differentiation.

Adipocyte differentiation of 3T3-L1 cells is a complex process which is inhibited by retinoic acid (RA). Since RA acts by nuclear receptors which directly regulate gene expression, we postulate that the primary targets of RA action in this system are genes which are regulated early in adipose conversion. In this study, we demonstrate the use of the differential display technique to search for early events in adipose commitment which are sensitive to RA. A mRNA was identified on the basis of its RA-dependent gene expression 24 h after initiation of a standard differentiation protocol. Molecular cloning of the cDNA revealed it to be identical to the ras recision gene (rrg), for lysyl oxidase. Indeed, two mRNAs identical to those recognized by lysyl oxidase probes were expressed in preadipocytes and tandemly repressed with 24 h of exposure to differentiation conditions. Lysyl oxidase activity was similarly reduced in the media of differentiated cells. RA completely blocked the differ-entiation-related reduction in rrg/lysyl oxidase gene expression, although RA had no independent stimulatory effect on rrg/lysyl oxidase expression in cells not exposed to differentiating conditions. Thus, differential display has been successfully used to identify rrg/lysyl oxidase as an early marker for adipose conversion that is responsive to RA.

3T3 Cells↗
PubMed1994

A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.

Amino Acid Sequence↗
PubMed1994

Neural network diagnosis of malignant melanoma from color images.

Malignant melanoma is the deadliest form of all skin cancers. Approximately 32,000 new cases of malignant melanoma were diagnosed in 1991 in the United States, with approximately 80% of patients expected to survive five years [1]. Fortunately, if detected early, even malignant melanoma may be treated successfully. Thus, in recent years, there has been rising interest in the automated detection and diagnosis of skin cancer, particularly malignant melanoma [2]. In this paper, we present a novel neural network approach for the automated separation of melanoma from three benign categories of tumors which exhibit melanoma-like characteristics. Our approach uses discriminant features, based on tumor shape and relative tumor color, that are supplied to an artificial neural network for classification of tumor images as malignant or benign. With this approach, for reasonably balanced training/testing sets, we are able to obtain above 80% correct classification of the malignant and benign tumors on real skin tumor images.

Adolescent↗
PubMed1994

Cloning of a complementary deoxyribonucleic acid encoding the murine homolog of the very low density lipoprotein/apolipoprotein-E receptor: expression pattern and assignment of the gene to mouse chromosome 19.

We report the cloning of a complementary DNA for the mouse homolog of the very low density lipoprotein (VLDL)/apolipoprotein-E receptor (VLDLR), the deduced amino acid sequence of the protein, and the mapping of the gene encoding the receptor to mouse chromosome 19. Northern hybridization revealed that the VLDLR messenger RNA (mRNA) is most abundant in skeletal muscle, heart, kidney, and brain. It was also detected in lung and in low levels in liver, but it was not found in spleen or testes. Levels of VLDLR mRNA in mouse placenta increased from days 8-18 of gestation. The VLDLR mRNA was induced in 3T3-L1 cells undergoing differentiation into adipocytes. The increase in VLDLR mRNA paralleled the rise in lipoprotein lipase and hormone-sensitive lipase mRNAs. However, VLDLR and low density lipoprotein receptor-related protein were increased in the presence of retinoic acid, whereas the induction of lipoprotein lipase and hormone-sensitive lipase mRNAs was inhibited. Our observations demonstrate regulated expression of the VLDLR gene in placenta and adipocytes, where the receptor protein may play roles in the uptake of triglyceride-rich particles for storage of lipid (adipocytes) or for lipid transport to the fetus (placenta). The availability of a murine complementary DNA probe and the knowledge of the map position of the VLDLR gene in the mouse genome will facilitate studies on the function and regulation of this protein.

3T3 Cells↗
PubMed1994

Peroxisome proliferator-activated receptor (PPAR) gamma: adipose-predominant expression and induction early in adipocyte differentiation.

Activators of peroxisome proliferator activated receptor (PPAR) regulate fatty acid metabolism and can induce adipocyte differentiation. We show here that the gamma subtype of PPAR is expressed at high levels in adipose tissue in contrast to a variety of other tissues, where little gene expression was noted. In addition, PPAR gamma is present at low levels in 3T3-L1 preadipocytes and is induced dramatically during adipocyte conversion using either normal differentiating conditions (fetal calf serum, dexamethasone, isobutyl-methylxanthine, and insulin) or the PPAR activator, WY-14,643. Thus PPAR gamma may be important for adipose cell development and function.

3T3 Cells↗
PubMed1993

Induction of Rev-ErbA alpha, an orphan receptor encoded on the opposite strand of the alpha-thyroid hormone receptor gene, during adipocyte differentiation.

Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related transcriptional activator that is encoded on the noncoding strand of the alpha-thyroid hormone receptor (TR) gene. The similarities between Rev-Erb and receptors for differentiating agents, as well as the abundance of Rev-Erb mRNA in fat, led us to study Rev-Erb gene expression during adipogenesis. Remarkably, Rev-Erb mRNA levels increased dramatically during the differentiation of 3T3-L1 cells into adipocytes. Rev-Erb was similarly induced in the related 3T3-F442A cell line but not in nondifferentiating 3T3-C2 cells. The time course of Rev-Erb induction was similar to that of C/EBP alpha, an important transcriptional regulator in adipocytes, and Rev-Erb mRNA was superinduced by cycloheximide. Nuclear run-on assays indicated that an increased rate of Rev-Erb mRNA synthesis accounted for the increased steady state mRNA levels; the half-life of Rev-Erb mRNA was indistinguishable in preadipocytes and adipocytes. Treatment of preadipocytes with retinoic acid inhibited adipocyte differentiation and also prevented Rev-Erb induction. Thus, there is a correlation between Rev-Erb gene expression and differentiation, and transcriptional regulation by Rev-Erb could play an important role in the generation and/or maintenance of the adipocyte phenotype. Interestingly, and possibly related to the overlap between the Rev-Erb gene and the exon specific for TR alpha 2, the induction of Rev-Erb was also associated with a 3-fold increase in the ratio of TR alpha 1 to TR alpha 2 mRNA levels, indicating that Rev-Erb expression has the potential to modulate adipocyte gene expression by multiple mechanisms.

3T3 Cells↗
PubMed1993

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.

Amino Acid Sequence↗
PubMed1993

Sustaining collaborative problem solving: strategies from a study in six Asian countries.

This paper presents findings from seven case studies of collaborative problem solving. The cases represent models of large-scale collaborative effort to improve the social, economic or health status of residents in poor communities. In these examples a variety of organizations worked together: government, business, voluntary agencies, community groups and people's organizations. A range of strategies was evident as these entities tried to sustain their collaboration. Strategies identified included those deployed collectively, those used by an individual organization to maintain its ability to participate and those used to persuade or pressure needed collaborators to become involved or stay the course. Specific strategies employed and descriptions of their use are presented and comprise a compelling profile of how collaborative problem solving functions.

Asia↗