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Biomedical subjects

M Merchant

Publications and source records attributed to M Merchant.

At least 19 recordsLinked to original sources
PubMed2001

LMP2A survival and developmental signals are transmitted through Btk-dependent and Btk-independent pathways.

The latent membrane protein 2A (LMP2A) of Epstein--Barr virus (EBV) has been implicated in controlling viral latency due to the ability of LMP2A to block B cell antigen receptor (BCR) signaling in vitro and to alter B cell development and enhance B cell survival in vivo. These LMP2A functions require interactions with the protein tyrosine kinases Syk and Lyn. However, a role for the Bruton's tyrosine kinase (Btk) has not been investigated for these LMP2A functions. To investigate whether Btk is important for LMP2A developmental and survival signals in vivo, LMP2A transgenic animals were mated to Btk deficient (Btk(-/-)) mice. Unlike LMP2A(+), Btk(+/+) transgenic littermate controls, LMP2A(+), Btk(-/-) animals do not generate immunoglobulin (Ig) receptorless B cells in the periphery and instead produce Ig(+) B cells similar to those in the Btk(-/-) mice. Interestingly, however, LMP2A(+), Btk(-/-) animals produce B cells at a vastly reduced level compared to Btk(-/-) littermates, indicating that LMP2A affects B cell development in the absence of Btk. In the RAG-1(-/-), Btk(-/-) double knockout background, LMP2A is still capable of enhancing the survival of Ig-receptorless B cells. Use of Btk phosphopeptide-specific antibodies reveals that Btk is constitutively phosphorylated in LMP2A-expressing cell lines. These data indicate that LMP2A initiates both Btk-dependent and Btk-independent pathways, resulting in altered B cell development and enhanced B cell survival.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2001

Epstein-Barr virus latent membrane protein 2A (LMP2A) employs the SLP-65 signaling module.

In latently infected B lymphocytes, the Epstein-Barr virus (EBV) suppresses signal transduction from the antigen receptor through expression of the integral latent membrane protein 2A (LMP2A). At the same time, LMP2A triggers B cell survival by a yet uncharacterized maintenance signal that is normally provided by the antigen receptor. The molecular mechanisms are unknown as LMP2A-regulated signaling cascades have not been described so far. Using a novel mouse model we have identified the intracellular adaptor protein Src homology 2 (SH2) domain-containing leukocyte protein (SLP)-65 as a critical downstream effector of LMP2A in vivo. Biochemical analysis of the underlying signaling pathways revealed that EBV infection causes constitutive tyrosine phosphorylation of one of the two SLP-65 isoforms and complex formation between SLP-65 and the protooncoprotein CrkL (CT10 regulator of kinase like). This leads to antigen receptor-independent phosphorylation of Cbl (Casitas B lineage lymphoma) and C3G. In contrast, phospholipase C-gamma2 (PLC-gamma2) activation is completely blocked. Our data show that in order to establish a latent EBV infection, LMP2A selectively activates or represses SLP-65-regulated signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The effects of the Epstein-Barr virus latent membrane protein 2A on B cell function.

Epstein-Barr Virus (EBV) infects B-lymphocytes circulating through the oral epithelium and establishes a lifelong latent infection in a subset of mature-memory B cells. In these latently infected B cells, EBV exhibits limited gene expression with the latent membrane protein 2A (LMP2A) being the most consistently detected transcript. This persistent expression, coupled with many studies ofthe function of LMP2A in vitro and invivo, indicates that LMP2A is functioning to control some aspect of viral latency. Establishment and maintenance of viral latency requires exquisite manipulation of normal B cell signaling and function. LMP2A is capable of blocking normal B cell signal transduction in vitro, suggesting that LMP2A may act to regulate lytic activation from latency in vivo. Furthermore, LMP2A is capable of providing B cells with survival signals in the absence of normal BCR signaling. These data show that LMP2A may help EBV-infected cells to persist in vivo. This review discusses the advances that have been made in our understanding of LMP2A and the effects it has on B cell development, activation, and viral latency.

Animals↗
PubMed2000

WW- and SH3-domain interactions with Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.

Amino Acid Sequence↗
PubMed2000

Recent advancements in surface-enhanced laser desorption/ionization-time of flight-mass spectrometry.

The overall history and recent advances in surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology is reviewed herein. Fundamentals of SELDI-TOF analysis are presented while drawing comparisons with other laser-based mass spectrometry techniques. The application of SELDI-TOF-MS to functional genomics and biomarker discovery is discussed and exemplified by elucidating a biomarker candidate for prostatic carcinoma. Finally, a short discussion regarding future SELDI requirements and developments is supplied.

Humans↗
PubMed2000

The LMP2A ITAM is essential for providing B cells with development and survival signals in vivo.

In Epstein-Barr virus-transformed B cells, known as lymphoblastoid cell lines (LCLs), LMP2A binds the tyrosine kinases Syk and Lyn, blocking B-cell receptor (BCR) signaling and viral lytic replication. SH2 domains in Syk mediate binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) in LMP2A. Mutation of the LMP2A ITAM in LCLs eliminates Syk binding and allows for full BCR signaling, thereby delineating the significance of the LMP2A-Syk interaction. In transgenic mice, LMP2A causes a developmental alteration characterized by a block in surface immunoglobulin rearrangement resulting in BCR-negative B cells. Normally B cells lacking cognate BCR are rapidly apoptosed; however, LMP2A transgenic B cells develop and survive without a BCR. When bred into the recombinase activating gene 1 null (RAG(-/-)) background, all LMP2A transgenic lines produce BCR-negative B cells that develop and survive in the periphery. These data indicate that LMP2A imparts developmental and survival signals to B cells in vivo. In this study, LMP2A ITAM mutant transgenic mice were generated to investigate whether the LMP2A ITAM is essential for the survival phenotype in vivo. LMP2A ITAM mutant B cells develop normally, although transgene expression is comparable to that in previously described nonmutated LMP2A transgenic B cells. Additionally, LMP2A ITAM mutant mice are unable to promote B-cell development or survival when bred into the RAG(-/-) background or when grown in methylcellulose containing interleukin-7. These data demonstrate that the LMP2A ITAM is required for LMP2A-mediated developmental and survival signals in vivo.

Amino Acid Sequence↗
PubMed1999

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

Angelman Syndrome↗
PubMed1998

Incidence of nosocomial pneumonia in a medical intensive care unit and general medical ward patients in a public hospital in Bombay, India.

We prospectively studied the incidence of hospital-acquired pneumonia in 1886 consecutive admissions to an 1800 bed hospital in Bombay; 991 of them to general medical wards and 895 to a 17-bed medical intensive care unit (ICU). The average bed occupancy in the general wards was 56 patients in a ward with 40 beds. Staffing in the general ward was two nurses for 56 patients, and in the ICU three nurses for 17 beds. One hundred and sixty-eight patients developed nosocomial pneumonia: 18 (1.8%) in general wards and 150 (16.7%) in the ICU. Common isolates included Pseudomonas spp (44%) and Klebsiella spp (34%). The most frequently used antibiotics were cefotaxime (34%), amikacin (25%), gentamicin (23%) and ofloxacin (13%). Crude mortality in general ward patients was 88.9 vs 14.6% in patients without pneumonia. The corresponding figures for ICU patients were 67.4 vs 37.1%; 40% of the crude mortality in ICU patients with pneumonia was attributable to the infection. Infected patients stayed an additional 5.8 days in the ICU and 6.7 days in the general ward. Costs of additional stay and antibiotics accounted for 18.6% of the ICU budget. The incidence of nosocomial pneumonia was lower than expected, despite occupancy exceeding bed capacity, low nurse:patient ratios, and extensive reuse of disposable respiratory therapy equipment. Nevertheless, nosocomial pneumonia imposes a significant financial burden on the already scarce resources available for intensive care in developing countries like India.

Adult↗
PubMed1996

Clinico-pathological features and survival of patients presenting with hepatic metastases: a retrospective analysis.

Retrospective analysis of 135 patients who presented with liver metastases at the NCI-Free Cancer Clinic and the Aga Khan University Hospital, Karachi is presented. The mean age was 55 years and there was no significant sexual predilection. Commonly observed primary sites were gall bladder (13%), pancreas (11%), breast (10%), colorectum (8%) and lung (8%). In 41 cases (30%), primary location remained undetermined. Most common histologic type was adenocarcinoma in both sexes. Majority (83%) had multiple metastases. Amongst those with solitary metastatis, 78% had right lobe involvement. Almost half of the patients presented with constitutional symptoms alone, while 31% had signs and symptoms related to hepatic involvement. Overall, the most commonly deranged laboratory tests were LDH (93%), albumin (84%), gamma glutamyl transferase (78%) and alkaline phosphatase (76%). Chemotherapy was offered to most cases. Majority of patients expired, mostly due to hepatic failure. Median survival was 30 days and was shorter in those who presented with multiple metastases. These findings are compatible with previous reports and may assist in the management of these patients.

Adult↗
PubMed1995

In vitro inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced activity by alpha-naphthoflavone and 6-methyl-1,3,8-trichlorodibenzofuran using an aryl hydrocarbon (Ah)-responsive construct.

Rat hepatoma H4IIE and mouse hepatoma Hepa 1c1c7 cells were transiently transfected with a plasmid construct that contained the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the mouse mammary tumor virus promoter and one copy of the dioxin responsive element. Treatment of transfected H4IIE and Hepa 1c1c7 cells with 10(-13) to 10(-6) M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a concentration-dependent increase in transient CAT activity. Maximum CAT activity was induced in both cell lines by exposure to 10(-9) M TCDD. The induction of CAT activity correlated well with the TCDD-induced, P4501A1-dependent ethoxyresorufin O-deethylase activity. Cotreatment of transfected cells with 10(-9) M TCDD and 10(-8) to 10(-6) M alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) resulted in a concentration-dependent reduction of TCDD-induced CAT activity. Treatment of cells with 10(-6) M alpha NF or MCDF alone resulted in only minimal induction of CAT activity. Both antagonists inhibited the induction of genes under the control of the CYP1A1 and mouse mammary tumor virus promoters, which indicates that the alpha NF- and MCDF-mediated antagonism of TCDD-induced, aryl hydrocarbon receptor-dependent gene transcription does not depend on promoter context.

Animals↗
PubMed1995

Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice.

Airway hyperresponsiveness is a key characteristic of human asthma and a marker for asthma-like conditions in animals. F1 mice derived from A/J and C57BL/6J display a phenotype which resembles the asthma-like phenotype of the A/J mice. Since airway responsiveness failed to segregate as a mendelian trait, we show significant linkage at two loci, Bhr1 (lod = 3.0) and Bhr2 (lod = 3.7) on chromosomes 2 and 15. A third locus, Bhr3 (lod = 2.83), maps to chromosome 17. Each of these loci maps near candidate loci implicated in the pathobiology of asthma. Our study represents the first linkages established through a genome-wide survey of airway hyperresponsiveness in any mammal.

Analysis of Variance↗
PubMed1994

Biphasic modulation of protein kinase C (PKC) activity by polychlorinated dibenzo-p-dioxins (PCDDs) in serum-deprived rat aortic smooth muscle cells.

Previous studies in this laboratory have shown that benzo(a)pyrene (BaP) modulates protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins. This observation is consistent with the ability of other aromatic hydrocarbons (AHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to modulate kinase activities in cells of hepatic, testicular, and thymic origin. Because all these chemicals share the ability to bind the aryl hydrocarbon receptor (AhR), the present studies were conducted to determine if changes in PKC activity by AHs conform with established structure-activity relationships. Experiments were conducted to examine the effects of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF), and 2,8-dichlorodibenzodioxin (DCDD) on the phosphorylation of exogenous histone type-III under basal and PKC-activating conditions. These congeners exhibit both high (TCDD and TCDF) and low (DCDD) AhR agonist activities. Measurements of kinase activity were conducted in the cytosolic and particulate fractions of growth-arrested (i.e., serum-deprived) cultured rat aortic SMCs incubated with 10 nM TCDD, TCDF, and DCDD for 0.5, 12, or 24 hours. No changes in basal kinase activity were induced by these chemicals at any of the times tested. Significant decreases in cytosolic and particulate PKC activity relative to controls were observed upon exposure of SMCs for 0.5 hours to 10 nM TCDD, TCDF, and DCDD. In contrast, SMCs exposed to TCDD and TCDF for 12 hours exhibited a significant increase in PKC activity in both cytosolic and particulate fractions. The PKC activity in cells exposed to DCDD for 12 hours was not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A genetic map of the mouse with 4,006 simple sequence length polymorphisms.

We have constructed a genetic map of the mouse genome containing 4,006 simple sequence length polymorphisms (SSLPs). The map provides an average spacing of 0.35 centiMorgans (cM) between markers, corresponding to about 750 kb. Approximately 90% of the genome lies within 1.1 cM of a marker and 99% lies within 2.2 cM. The markers have an average polymorphism rate of 50% in crosses between laboratory strains. The markers are distributed in a relatively uniform fashion across the genome, although some deviations from randomness can be detected. In particular, there is a significant underrepresentation of markers on the X chromosome. This map represents the two-thirds point toward our goal of developing a mouse genetic map containing 6,000 SSLPs.

Animals↗
PubMed1993

Modulation of protooncogene expression in rat aortic smooth muscle cells by benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), cause vascular lesions of atherosclerotic etiology in avian and rodent species. Because the development of these lesions is associated with aberrant proliferation of vascular smooth muscle cells (SMCs), the present study was conducted to evaluate the effects of BaP on DNA synthesis and protooncogene expression in rat aortic SMCs. Subcultured cells were exposed to BaP (0.3-30 microM) for various times and processed for measurements of [3H]thymidine incorporation and c-Ha-ras or c-myc protooncogene expression. Sucrose density gradient analysis and gel mobility shift assays were employed to assess binding of BaP to intracellular proteins in the nuclear fraction and their interaction with a synthetic dioxin responsive element (DRE), respectively. BaP (0.3 and 3 microM) increased DNA synthetic rates in randomly cycling SMCs in a concentration- and time-dependent fashion. Increased DNA synthesis was also observed in synchronized SMCs treated with 3.0 microM BaP. Challenge of quiescent SMCs with 10% fetal bovine serum in the presence of BaP for 8 h enhanced serum-induced c-Ha-ras and c-myc protooncogene expression. Velocity sedimentation analysis of the nuclear fraction from SMCs treated with 0.3 microM [3H]BaP resulted in specifically bound peaks of 4.4 S and 6.5 S. The 6.5 S peak was competitively inhibited in the presence of unlabeled 10 microM 2,3,7,8-tetrachlorodibenzofuran, an aryl hydrocarbon ligand, while both peaks were eliminated in cells cotreated with 10 microM alpha-naphthoflavone (alpha-NF), an inhibitor of cytochrome P450 and an antagonist of PAH-protein interactions. alpha-NF also inhibited the induction of c-Ha-ras mRNA and DNA synthesis by BaP and the binding of BaP-Ah receptor complexes to the DRE. These results show that BaP enhances serum-induced protooncogene expression during the early part of the cell cycle in rat aortic SMCs and suggest that binding of BaP to intracellular proteins may play a role in these responses.

Animals↗
PubMed1993

Mechanism of action of alpha-naphthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells.

alpha-Naphthoflavone (alpha NF) and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 gene expression in MCF-7 human breast cancer cells and also decreased the accumulation of the nuclear [3H]TCDD-aryl hydrocarbon (Ah) receptor complex. Nuclear extracts from cells treated with 10(-6) M alpha NF and incubated with a dioxin responsive element (DRE, 26-mer) did not form a retarded band in a gel mobility shift assay. In contrast, incubation of nuclear extracts from cells treated with 10(-6) M MCDF and DRE gave a retarded band and this is consistent with the antiestrogenic and Ah receptor agonist activity of MCDF in human breast cancer cells. alpha NF was further investigated as an Ah receptor antagonist by determining the inhibition by alpha NF of TCDD-induced antiestrogenicity in MCF-7 cells. TCDD (10(-9) M) inhibited the 17 beta-estradiol-induced proliferation of MCF-7 cells and the secretion of the 52-kDa protein. In cotreatment studies, alpha NF (10(-8) to 10(-6) M) caused a concentration-dependent decrease in the antiestrogenic responses elicited by TCDD. In addition, alpha NF inhibited the TCDD-induced down-regulation of nuclear estrogen receptor levels in MCF-7 cells. alpha NF (10(-6) M) alone was inactive as an estrogen or antiestrogen and in cotreatment studies did not affect 17 beta-estradiol-induced responses in MCF-7 cells. Tamoxifen (10(-7) M), an antiestrogen which acts through the estrogen receptor, also inhibited 17 beta-estradiol-induced cell proliferation and alpha NF did not affect the tamoxifen-mediated antiproliferative response. Thus, alpha NF antagonized TCDD-induced CYP1A1 gene expression in MCF-7 cells and also acted as an anti-antiestrogen for TCDD-mediated antiestrogenicity in these cells. These results were consistent with the low levels of DRE binding observed with nuclear extracts from cells treated with 10(-9) M TCDD plus alpha NF (10(-8) to 10(-6) M) or 10(-6) M alpha NF alone. Thus, alpha NF appears to act as an Ah receptor antagonist in MCF-7 cells by decreasing the levels of transcriptionally active nuclear Ah receptor complexes.

Benzoflavones↗
PubMed1993

An unusual case of retroperitoneal fibrosis.

With several hundred cases reported there is still much speculation about the etiology of idiopathic retroperitoneal fibrosis. We report an unusual case of retroperitoneal fibrosis with histologically identical lesion in the maxillary sinus, which has not been previously described. Both lesions showed dramatic response to corticosteroids but relapsed frequently on withdrawal of steroids, highlighting the importance of long-term follow-up.

Acute Kidney Injury↗
PubMed1993

Planning for high risk ophthalmic ambulatory patients.

Ophthalmic ambulatory care settings are faced with providing expert care for a high volume of individuals with increasingly complex nursing and psychosocial needs. These needs may include financial assistance, medications, transportation, shelter, home healthcare, and various community resources. A case study will demonstrate the effectiveness of interdisciplinary collaboration between nursing and social services in identifying high risk ophthalmic patients, coordinating resources, and decreasing fragmentation of care.

Aged↗