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Biomedical subjects

A Chawla

Publications and source records attributed to A Chawla.

At least 19 recordsLinked to original sources
PubMed2001

Increased engraftment and GVHD after in utero transplantation of MHC-mismatched bone marrow cells and CD80low, CD86(-) dendritic cells in a fetal mouse model.

BACKGROUND: Bone marrow transplantation (BMT) is the only known cure for a variety of inherited diseases and requires the administration of high doses of immunosuppressive and myeloablative therapy. Because the fetus is immunoincompetent early in gestation, in utero stem cell transplantation (IUT) could avoid the need for this toxic conditioning. A major limitation to date of IUT is the low level of engraftment and failure to induce tolerance. Dendritic cells (DC) are considered very potent antigen-presenting cells, but DC progenitors (pDC) are strongly tolerogenic. METHODS: We examined the effect of donor pDC on the degree of engraftment and tolerance induction after IUT. Bone marrow-derived pDC (CD80low, CD86-) from male C57BL/6 mice (H2b) were injected with and without donor bone marrow (BM) intraperitoneally into 13 to 15-day BALB/c (H2d) fetuses. Engraftment was determined by flow cytometry and quantitative polymerase chain reaction and tolerance by skin grafts and the mixed lymphocyte reaction. RESULTS: At 1-month posttransplant, mice that received BM+pDC showed a higher degree of engraftment (29+/-46%) than mice that received pDC-enriched cells or BM cells alone (0.11+/-0.70% and 1.71+/-1.66%, respectively, P<0.001). However, 5/19 recipients of BM+pDC died within 6 weeks; 4/5 had significant donor cell engraftment in blood and/or tissues. Also, these mice had evidence of graft-versus-host disease (GVHD). Two mice out of 15 long-term survivors in the BM+pDC group had virtually complete replacement of host with donor hematopoietic cells. Skin grafts and mixed lymphocyte reaction studies showed no durable tolerance induction other than in the two fully engrafted recipients of BM+pDC. CONCLUSIONS: These results suggest that donor pDC, along with donor BM, can have a significant impact on engraftment of MHC-mismatched donor cells associated with an increased incidence of GVHD. However, marrow-derived pDC do not result in an increase in tolerance induction in utero even when microchimerism is present.

Animals↗
PubMed2001

Nuclear receptors and lipid physiology: opening the X-files.

Cholesterol, fatty acids, fat-soluble vitamins, and other lipids present in our diets are not only nutritionally important but serve as precursors for ligands that bind to receptors in the nucleus. To become biologically active, these lipids must first be absorbed by the intestine and transformed by metabolic enzymes before they are delivered to their sites of action in the body. Ultimately, the lipids must be eliminated to maintain a normal physiological state. The need to coordinate this entire lipid-based metabolic signaling cascade raises important questions regarding the mechanisms that govern these pathways. Specifically, what is the nature of communication between these bioactive lipids and their receptors, binding proteins, transporters, and metabolizing enzymes that links them physiologically and speaks to a higher level of metabolic control? Some general principles that govern the actions of this class of bioactive lipids and their nuclear receptors are considered here, and the scheme that emerges reveals a complex molecular script at work.

Animals↗
PubMed2001

Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The absence of culture systems permissive for HCV replication has presented a major bottleneck to antiviral development. We sought to recapitulate the early steps in the life cycle of HCV by means of DNA-based expression of viral genomic sequences. Here we report expression of replicating HCV RNA by using a, to our knowledge, novel binary expression system in which cells were transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis Delta ribozyme to control 3' cleavage, and infected with vaccinia-T7 polymerase. HCV genomic and replicative strand synthesis, in addition to protein synthesis, was detectable and depended on full-length HCV sequences. Moreover, the system was capable of generating HCV RNA quasispecies, consistent with the action of the low-fidelity HCV NS5B RNA polymerase. IFN-alpha, but not ribavirin, directly inhibited the viral replicative cycle in these cells, identifying the virus itself and not solely the immune system as a direct target of IFN action. The availability of a cell-based test for viral replication will facilitate screening of inhibitory compounds, analysis of IFN-resistance mechanisms, and analysis of virus-host cell interactions.

Amantadine↗
PubMed2001

Signaling complexes of the FERM domain-containing protein GRSP1 bound to ARF exchange factor GRP1.

GRP1 is a member of a family of proteins that contain a coiled-coil region, a Sec7 homology domain with guanosine nucleotide exchange activity for the ARF GTP-binding proteins, and a pleckstrin homology domain at the C terminus. The pleckstrin homology domain of GRP1 binds phosphatidylinositol (3,4,5) trisphosphate and mediates the translocation of GRP1 to the plasma membrane upon agonist stimulation of PI 3-kinase activity. Using a (32)P-labeled GRP1 probe to screen a mouse brain cDNA expression library, we isolated a cDNA clone encoding a GRP1-binding partner (GRSP1) that exists as two different splice variants in brain and lung. The GRSP1 protein contains a FERM protein interaction domain as well as two coiled coil domains and may therefore function as a scaffolding protein. Mapping experiments revealed that the interaction of GRP1 and GRSP1 occurs through the coiled coil domains in the two proteins. Immunodepletion experiments indicate that virtually all of the endogenous GRSP1 protein exists as a complex with GRP1 in lung. When co-expressed in Chinese hamster ovary cells expressing the human insulin receptor, both proteins display a diffuse, cytoplasmic localization. Acute translocation and co-localization of GRSP1 and GRP1 to ruffles in the plasma membrane was evident after insulin stimulation. These results identify GRSP1 as a novel member of GRP1 signaling complexes that are acutely recruited to plasma membrane ruffles in response to insulin receptor signaling.

ADP-Ribosylation Factors↗
PubMed2001

A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis.

Previous work has implicated PPAR gamma in the regulation of CD36 expression and macrophage uptake of oxidized LDL (oxLDL). We provide evidence here that in addition to lipid uptake, PPAR gamma regulates a pathway of cholesterol efflux. PPAR gamma induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by the nuclear receptor LXR alpha. Ligand activation of PPAR gamma leads to primary induction of LXR alpha and to coupled induction of ABCA1. Transplantation of PPAR gamma null bone marrow into LDLR -/- mice results in a significant increase in atherosclerosis, consistent with the hypothesis that regulation of LXR alpha and ABCA1 expression is protective in vivo. Thus, we propose that PPAR gamma coordinates a complex physiologic response to oxLDL that involves particle uptake, processing, and cholesterol removal through ABCA1.

ATP Binding Cassette Transporter 1↗
PubMed2001

Autonomic dysfunction and cholelithiasis in patients with cirrhosis.

Gallstones are seen in 33-46% of patients with cirrhosis, and their prevalence is known to increase with the duration and severity of liver disease. We hypothesized that autonomic neuropathy may contribute to the formation of gallstones or gallbladder disease, as in diabetics with autonomic neuropathy, due to impaired gallbladder emptying. The objective of our study was to determine the prevalence of gallstones or gallbladder disease in cirrhotic patients with and without autonomic neuropathy. We determined autonomic function tests, gallstones, and other gallbladder disease in 123 (male 71) with varying severity of liver disease (Child classes: A, 40; B, 45; C, 35). In all, 54 patients had gallstones and an additional 22 patients had other gallbladder disease (cholecystitis, common bile duct stones, or debris). Autonomic neuropathy was seen in 97 patients (one abnormal test in 48 and two or more in 49). The prevalence of gallstones was similar in Child A (57%), Child B (64%), and Child C (63%) cirrhosis. The gallstones or gallbladder disease was not increased in women, blacks, diabetics, or alcoholic cirrhotics. The prevalence of gallbladder disease was increased in patients with autonomic neuropathy (51% vs 35%, P = 0.08); in patients with Child C cirrhosis, gallstones (P = 0.018) and gallbladder disease (P = 0.03) were seen more commonly in patients with autonomic neuropathy. Our findings suggest that autonomic neuropathy may contribute to the formation of gallstones in patients with advanced cirrhosis, perhaps by impairing gallbladder and sphincter of Oddi dysmotility.

Adult↗
PubMed2001

PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is highly expressed in lipid-accumulating macrophages of the coronary artery. In light of this, the wide-spread clinical use of thiazolidinediones (TZDs) in the treatment of type II diabetes raises concerns about the role of PPAR-gamma in macrophage function and disease progression. To define the role of PPAR-gamma in macrophage biology, we used homologous recombination to create embryonic stem cells that were homozygous for a null mutation in the PPAR-gamma gene. We demonstrate here that PPAR-gamma is neither essential for nor substantially affects the development of the macrophage lineage both in vitro and in vivo. In contrast, we show it is an important regulator of the scavenger receptor CD36, which has been genetically linked to lipid accumulation in macrophages. Both 15-deoxy-Delta12,14prostaglandin J2 and thiazolidinediones have anti-inflammatory effects that are independent of PPAR-gamma. We show that PPAR-gamma is required for positive effects of its ligands in modulating macrophage lipid metabolism, but that inhibitory effects on cytokine production and inflammation may be receptor independent.

3T3 Cells↗
PubMed2001

Insulin action on GLUT4 traffic visualized in single 3T3-l1 adipocytes by using ultra-fast microscopy.

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20-30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5-7 min at 37 degrees C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.

3T3 Cells↗
PubMed2001

G(alpha)11 signaling through ARF6 regulates F-actin mobilization and GLUT4 glucose transporter translocation to the plasma membrane.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

ADP-Ribosylation Factor 6↗
PubMed2000

Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs.

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)), with K(D) values of 0.05, 1.6 and 1.0 micrometer for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4, 5)P(3) versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly(274)-Gly(275) motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P(2) with little effect on its binding to PtdIns(3,4,5)P(3), while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P(2) but not for PtdIns(3,4,5)P(3). In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P(3) binding selectivity.

Amino Acid Sequence↗
PubMed2000

A role for phospholipase D in GLUT4 glucose transporter translocation.

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitope-tagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.

3T3 Cells↗
PubMed2000

Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains.

Lipid second messengers generated by phosphoinositide (PI) 3-kinases regulate diverse cellular functions through interaction with pleckstrin homology (PH) domains in modular signaling proteins. The PH domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds phosphatidylinositol 3,4,5-trisphosphate with high affinity. We have determined the structure of the Grp1 PH domain in the unliganded form and bound to inositol 1,3,4,5-tetraphosphate. A novel mode of phosphoinositide recognition involving a 20-residue insertion within the beta6/beta7 loop explains the unusually high specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains including that of protein kinase B. When compared to other PH domains, general determinants of 3-phosphoinositide recognition and specificity can be deduced.

Amino Acid Sequence↗
PubMed2000

Inflammatory polyps after necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in the neonatal period. NEC causes ulceration of the intestinal mucosa and may lead to perforation or a stricture. To the best of the authors' knowledge intestinal inflammatory polyps after NEC have not been described previously. The authors report on a 17-week-old boy with pseudopolyps at the site of a colonic stricture after NEC.

Colonic Polyps↗
PubMed1999

ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.

3T3 Cells↗
PubMed1998

HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes.

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes. Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has not been known. We now show that repression results from the interaction of Tat with the TAFII250 component of the general transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat, including the MHC class I promoter, are dependent on TAFII250 whereas those that are not repressed by Tat, such as SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription would be one mechanism by which HIV avoids immune surveillance.

Acetyltransferases↗