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Biomedical subjects

P Banerjee

Publications and source records attributed to P Banerjee.

At least 55 records · Page 3Linked to original sources
PubMed1999

MHC class II alpha/beta heterodimeric cell surface molecules expressed from a single proviral genome.

Transplantation tolerance to renal allografts can be induced in large animal preclinical models if the donor and recipient have identical major histocompatibility complex (MHC) class II loci. Such class II matching is, however, not clinically achievable owing to the extreme diversity of class II sequences. With the ultimate goal of creating a somatic class II match in the bone marrow of an allograft recipient, the aim of the study is to develop a double-copy retrovirus construct to express both chains of the MHC class II DQ glycoprotein on a single transduced cell. Analysis of the expression patterns of the retroviral DQ transgenes in both virus producer and transduced fibroblasts revealed correct transcription and stable surface expression of the DQ heterodimers. In addition, we demonstrate that both the DQA and DQB sequences are functional within the same proviral copy, a prerequisite for efficient induction of transplantation tolerance following transduction of bone marrow precursor cells. The DQ double-copy retrovirus vector showed efficient expression of the transferred class II cDNA in murine colony-forming units for the granulocyte-monocyte lineage (CFU-GM), indicating that it is suitable for gene therapy of multimeric proteins in hematopoietic cells.

3T3 Cells↗
PubMed1999

cbl-3: a new mammalian cbl family protein.

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed1999

cbl-b inhibits EGF-receptor-induced apoptosis by enhancing ubiquitination and degradation of activated receptors.

Studies in C. elegans and Drosophila melanogaster suggest that cbl proteins are inhibitors of epidermal growth factor receptor (EGFR) function. Here we describe that overexpression of cbl-b, a homologue of the c-cbl protooncogene, inhibits EGFR-induced apoptosis in MDA-MB-468 breast cancer cells. Overexpression of cbl-b results in a shortened duration of EGFR activation upon EGF stimulation. This is demonstrated by decreased amounts of phosphorylated EGFR as well as by inhibition of multiple downstream signaling pathways. The inhibition of signaling by cbl-b results from increased ubiquitination and degradation of the activated EGFR. The inhibitory effects of cbl-b overexpression on apoptosis and on EGFR signaling are reversed by blocking proteosomal degradation of the EGFR. These data demonstrate that the mechanism by which cbl-b inhibits EGFR-induced apoptosis is by activation-dependent degradation of the EGFR. They imply that this mechanism may be a general one whereby cbl proteins regulate intracellular signaling.

Acetylcysteine↗
PubMed1999

Agonist stimulation of the serotonin1A receptor causes suppression of anoxia-induced apoptosis via mitogen-activated protein kinase in neuronal HN2-5 cells.

Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin1A receptor (5-HT1A-R), could cause attenuation of the activity of both N-type Ca2+ channels and N-methyl-D-aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5-HT1A-R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca2+ channels. Using a hippocampal neuron-derived cell line (HN2-5) that is Ca2+ channel-deficient, we demonstrate here that an alternative pathway is responsible for 5-HT1A-R-mediated protection of these cells from anoxia-triggered apoptosis, assessed by deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL). The 5-HT1A-R agonist-evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation-mediated activation of mitogen-activated protein kinase (MAPK), as evidenced by the elimination of the agonist-elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI-3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5-HT1A-R caused a 60% inhibition of anoxia-stimulated caspase 3-like activity in the HN2-5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5-HT1A-R caused an activation of PI-3Kgamma in HN2-5 cells, our results showed that this PI-3Kgamma activity was not linked to the 5-HT1A-R-promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2-5 cells, agonist binding to the 5-HT1A-R results in MAPK-mediated inhibition of a caspase 3-like enzyme and a 60-70% suppression of anoxia-induced apoptosis through a Ca2+ channel-independent pathway.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1999

Interaction between fluconazole and amphotericin B in mice with systemic infection due to fluconazole-susceptible or -resistant strains of Candida albicans.

The interaction between fluconazole (Flu) and amphotericin B (AmB) was evaluated in a murine model of systemic candidiasis for one Flu-susceptible strain (MIC, 0.5 microg/ml), two strains with intermediate Flu resistance (Flu mid-resistant strains) (MIC, 64 and 128 microg/ml), and one highly Flu-resistant strain (MIC, 512 microg/ml) of Candida albicans. Differences in fungal densities in kidneys of infected mice after 24 h of therapy and in survival rates at 62 days of mice treated with an antifungal drug or a combination of antifungal drugs for 4 days were compared. For the Flu-susceptible and Flu mid-resistant strains, the combination of Flu and AmB was antagonistic, as shown by both quantitative culture results and survival. The interaction was additive for the highly Flu-resistant strain. These results suggest that the combination of Flu and AmB should be used with caution in infections due to fungi that are usually susceptible to both antifungal agents and as empirical antifungal drug therapy.

Amphotericin B↗
PubMed1999

Tubby-like protein 1 homozygous splice-site mutation causes early-onset severe retinal degeneration.

PURPOSE: To characterize the disease expression of an autosomal recessive human retinal degeneration associated with a mutation in TULP1 (tubby-like protein 1), a gene with currently unknown function. METHODS: Homozygotes and heterozygotes from an extended Dominican kindred with a TULP1 splice-site gene mutation (IVS14+1,G-->A) were studied clinically and with visual function tests. Sequence analysis of TULP1 was also performed in unrelated patients with severe retinal degeneration from a North American clinic population. RESULTS: Homozygotes had nystagmus, visual acuity of 20/200 or worse, color vision disturbances, bull's eye maculopathy, and peripheral pigmentary retinopathy. Younger patients had a relatively wide extent of kinetic visual fields; older patients had only peripheral islands. No rod function was measurable by psychophysics in any of the patients; markedly reduced cone function was detectable across the visual field of younger patients and in the remaining peripheral islands of older patients. Rod and cone electroretinograms (ERGs) were not detectable using standard methods; microvolt-level cone ERGs were present in some patients. Heterozygotes had normal visual function. No putative pathogenic sequence changes in TULP1 were observed in North American patients with comparably severe retinal phenotypes, mainly in the diagnostic category of Leber congenital amaurosis. CONCLUSIONS: This TULP1 splice-site mutation in homozygotes causes early-onset, severe retinal degeneration involving macular and peripheral cones and rods. The constellation of phenotypic findings suggests that the TULP1 gene product is critically important for normal photoreceptor function and may play a role in retinal development.

Adolescent↗
PubMed1998

Homozygosity and physical mapping of the autosomal recessive retinitis pigmentosa locus (RP14) on chromosome 6p21.3.

Retinitis pigmentosa (RP) is a heterogeneous genetic disorder with autosomal dominant, autosomal recessive, and X-linked forms. We previously mapped an additional arRP locus to chromosome 6p21 (RP14) in a single extended kinship from the Dominican Republic. Aided by a second linked RP pedigree from the same region of the Dominican Republic, we have refined the disease locus to a 2-cM region that is homozygous-by-descent in both pedigrees. A complete YAC, and a partial BAC, contig of the RP14 locus was constructed between the markers D6S1560 and D6S291, encompassing approximately 2.1 Mb. The contig contains 12 YACs and 31 BACs and is characterized by 45 markers including 8 microsatellite markers, 6 gene-derived sequences/ESTs obtained from the databases, and 28 new STSs and 4 new ESTs obtained by BLAST search using DNA sequence from the ends of the BAC and YAC inserts. With a STS density of approximately 1 every 20 kilobases, this contig significantly enhances available maps of the region.

Chromosome Mapping↗
PubMed1998

TULP1 mutation in two extended Dominican kindreds with autosomal recessive retinitis pigmentosa.

The RP14 autosomal recessive Retinitis pigmentosa (arRP) locus has been mapped to a 2cM region of chromosome 6p21.3. TULP1 (the gene encoding tubby-like protein 1) is a candidate target for the disease mutation because it maps to the RP14 minimum genetic region and because a mutation in the highly homologous mouse tub gene leads to obesity, deafness and early progressive retinal degeneration. Here we report a splice-site mutation (IVS14+1, G-->A) that is homozygous in all affected individuals (N=33) and heterozygous in all obligate carriers (N=50) from two RP14-linked kindreds. The mutation was not observed in 210 unrelated controls. The data indicate that impairment of TULP1 protein function is a rare cause of arRP and that the normal protein plays an essential role in the physiology of the retina.

Animals↗
PubMed1998

Changes in CD23 expression of blood and skin in atopic eczema after Chinese herbal therapy.

BACKGROUND: Aberrant expression of CD23 (low affinity IgE receptor) on cells of the monocyte/macrophage series in peripheral blood and lesional skin of patients with atopic eczema has been demonstrated. It is not known whether this abnormality results from a fundamental systemic problem of the monocytes of these patients or reflects local changes to cell populations within the skin tissues. OBJECTIVES: This study was designed to determine whether this aberrant expression was caused by local cutaneous influences on mature cells or fundamental changes in monocyte differentiation. The possible relationship between these aberrations and clinical severity was also investigated by repeating these immunopathological studies after a course of efficacious treatment with Chinese herbal therapy (CHT). METHODS: Peripheral blood mononuclear cells were obtained from patients with atopic eczema before, and after 8 weeks of treatment. Efficacy of CHT was quantified on clinical grounds. Monocytes were isolated by adherence to plastic and cultured for up to 7 days. Samples were harvested at 2, 5 and 7 days of culture and cytospins prepared. Immunocytochemical staining to identify phenotypic subsets was performed on the monocytes at time 0 and on maturing cells from culture. This immunocytology was quantified using computerized image analysis equipment to determine the emergence of macrophage subsets and their level of CD23 expression. Biopsies were taken from lesional skin before and after treatment and immunohistology was performed on cryostat sections to determine the number of antigen presenting cells expressing CD23 as well as the level of expression of these molecules. RESULTS: The results showed that increased numbers of monocytes from patients with atopic eczema express CD23 at day 0 and that cultured monocytes from these patients differentiate faster during the 7 day culture period as compared to normal controls. Efficacious treatment did not affect the number of peripheral blood monocytes expressing CD23. However, treatment did lead to a significant decrease in the number of CD23+ mature macrophages in the skin as well as a reduction in the level of expression of this moiety. These results demonstrate that changes in clinical severity are more closely related to the expression of CD23 on mature antigen presenting cells in lesional skin rather than to differentiating peripheral blood monocyte CD23 expression. CONCLUSIONS: These results suggests that local factors within lesional skin govern the accumulation and the expression of CD23 on mature macrophages and that these factors may be more relevant to the pathogenesis of the disease than aberrations in CD23 expression that may occur systemically.

Adolescent↗
PubMed1998

Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells, but only the phosphatidylserine-displaying apoptotic cells are phagocytosed by microglia.

Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS-derived neuronal cells was tested using hippocampal HN2-5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum-containing medium, did not reattach, and (c) cells that reattached in 2-3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide-positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate-annexin V-positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a-c also showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS-derived HN2-5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.

Amoeba↗
PubMed1998

Pharmacodynamics of fluconazole in a murine model of systemic candidiasis.

In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated Candida albicans infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED50]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED50 of fluconazole given intraperitoneally was 4.56 mg/kg of body weight/day (95% confidence interval, 3.60 to 5.53 mg/kg/day), and the dose-response relationship was best described by an inhibitory sigmoid maximal effect (Emax) curve. To define the pharmacodynamics of fluconazole, we gave dosages lower than, approximating, and higher than the ED50 of fluconazole (range, 3.5 to 5.5 mg/kg/day, equivalent to the ED16 to the ED75) to various groups of infected animals using three dose-fractionation schedules. For each total dose of fluconazole examined, the dose-fractionation schedules optimized the ratio of the area under the concentration-time curve (AUC) to the MIC (the AUC/MIC ratio), the ratio of the maximum concentration of drug in serum (Cmax) to the MIC, and the time that the drug remained above the MIC for the infecting C. albicans isolate. Similar reductions in fungal densities in kidneys were seen between groups that received the same total dose of fluconazole in one, two, or four equally divided doses. Thus, dose-fractionation studies demonstrated that the pharmacodynamic parameter of fluconazole that best predicted outcome was the AUC/MIC ratio.

Animals↗
PubMed1998

Identification of a full-length cDNA for an endogenous retrovirus of miniature swine.

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attachment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.

Amino Acid Sequence↗
PubMed1997

The human GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on the short arm of chromosome 6 (p21.1).

GCAP1 and GCAP2 are related Ca(2+)-binding proteins that activate photoreceptor guanylate cyclase(s). We showed previously that the human GCAP1 gene, consisting of four exons, is located at 6p21.1 (locus designation GUCA). To identify the chromosomal location of the GCAP2 gene, we first cloned its cDNA and determined its intron-exon distribution by PCR analysis. The results show that the introns of the GCAP2 gene are positioned exactly as in the GCAP1 gene and are nearly double in size. Sequence similarity between the two genes, however, is limited to portions of exons 1 and 2. The GCAP1 and GCAP2 genes are transcribed into single mRNA species (1.7 and 2.2 kb, respectively) and are detectable only in the retina by Northern blotting. The GCAP2 gene was found by somatic human-hamster hybrid panel analysis and FISH to reside at GUCA in a region indistinguishable from that of GCAP1. PCR analysis with exon 4-specific primers showed that the genes are in a tail-to-tail array less than 5 kb apart and altogether span less than 20 kb of genomic DNA. The identical gene structures and loci of GCAP1 and GCAP2, and the identical function of the gene products, are consistent with gene duplication event.

Amino Acid Sequence↗
PubMed1997

Modulation by Chinese herbal therapy of immune mechanisms in the skin of patients with atopic eczema.

Ten patients with atopic eczema (AE) received treatment with Chinese herbal therapy (CHT; Zemaphyte) for 2 months. The severity of the eczema was recorded and skin biopsies were taken from lesional (L) and non-lesional (NL) skin before and after treatment. The skin biopsies were stained to detect T-cell subsets (CD4, CD8, CD45Ro and CD25), macrophage subsets (RFD7), dendritic cells (RFD1). Langerhans cells (CD1), HLA-DR, low-affinity IgE receptors (CD23) and high-affinity IgE receptors (15A5, 22H7). A quantitative assessment of the numbers of positively stained cells was made. Monoclonal antibody binding specifically to CD23(Fc epsilon RII) was used, in combination with cell subset monoclonal antibodies to quantify the cellular distribution of CD23 antigen in the skin. Following 2 months of treatment with CHT, erythema was reduced by 53%. There was also a significant reduction in HLA-DR expression. The numbers of RFD1 + CD23 +, RFD7 + CD23 +, CD1 + CD23+ and CD25 + cells in lesional skin decreased significantly after treatment (RFD1 + CD23 + from 0.39 to 0.21, RFD7 + CD23 + from 0.29 to 0.16. CD1 + CD23 + from 0.24 to 0.09, CD25 + from 0.84 to 0.31 in epidermis and from 1.62 to 0.94 in dermis (mean cells numbers per unit area). No significant change in cell numbers in NL skin or expression of Fc epsilon RI in either L or NL samples was observed after treatment. This study confirms that CHT is clinically efficacious and that clinical improvement is associated with a significant reduction in antigen-presenting cells expressing CD23.

Adult↗
PubMed1996

Apoptosis is associated with an increase in saturated fatty acid containing phospholipids in the neuronal cell line, HN2-5.

Two widely different paradigms of stress, hypoxia and nutrient deprivation, were observed to trigger apoptosis in a clonal neuronal (hippocampal) cell line (HN2-5) as judged by DNA laddering analysis and chromatin condensation. Since according to its original definition, apoptosis is concomitant with a dramatic change in cell morphology, the composition of major phospholipids that determine morphological properties of cells was analyzed in this study. We observed a significant increase (10-20%) in the proportion of saturated fatty acid side chains only in phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) but not in phosphatidylcholine (PC). This increase was independent of the paradigm of stress used (hypoxia or nutrient deprivation) to initiate apoptosis. Since phospholipids constitute the fabric of both plasma membranes as well as the membranes surrounding the intracellular organelles such as the lysosomes, endoplasmic reticulum and the nuclei, such an apoptosis-associated, selective change in phospholipid composition could cause a dramatic and overall change in membrane fluidity, thus producing the typical shrinking, deformation and porosity of both nuclear as well as plasma and lysosomal membranes as observed in apoptotic cells. Leakiness of lysosomal and nuclear membranes could cause diffusion of deoxyribonucleases into cell nuclei, thus resulting in the characteristic cleavage and laddering of chromosomal DNA which accompany apoptosis.

Antibodies↗
PubMed1996

Cell-specific regulation of the stably expressed serotonin 5-HT1A receptor and altered ganglioside synthesis.

Neurotransmission is dependent on the presence of neuronal receptors at the synapses, and important cell surface molecules such as gangliosides are pivotal in the maintenance of synaptic contacts. To study the interrelationship between these two classes of molecules, we achieved stable expression of the hippocampus- and CNS-localized serotonin 1A receptor (5-HT1A-R) in three 5-HT1A-R-deficient neuronal cell lines and also the control, non-neural CHO cells. A strong passage dependence of 5-HT1A-R expression, as measured by mRNA levels as well as membrane binding to the selective agonist [3H]8-OH-DPAT, was observed only in the HN2 (hippocampal) and NCB-20 (CNS) cells which are derived from tissues of natural occurrence of the 5-HT1A-R. A paradigm of stress was obtained by carrying out continuous culture of cells without feeding. During this time a dramatic increase in 5-HT1A-R mRNA and [3H]8-OH-DPAT binding was observed only in the neuronal cells after confluence and during decreased cell viability (days 10/11). This was not due to differentiation, since deliberate serum deprivation and differentiation of cells did not result in any dramatic increase in 5-HT1A-R expression. Analysis of ganglioside synthesis by pulse labeling of the transfected cells produced striking results. In the dorsal root of the ganglion (DRG) derived F-11 cells which show low but significant levels of complex gangliosides before transfection, the mere presence of the serotonin 1A receptor resulted in a dramatic increase in synthesis of gangliosides comigrating with GM2, GD1a, GD1b, and GT1b (20-fold by densitometry). In contrast, there was only a 2-fold increase in the overall content of complex gangliosides in the presence of the 5-HT1A-R. In the NCB-20 cells which contain only GD1a but no GD1b or GT1b before transfection, a decrease in GD1a synthesis was observed following transfection. Also agonist (8-OH-DPAT) binding to the serotonin 1A receptor in NCB-20 cells produced a 3-fold increase in synthesis of a ganglioside comigrating with GM3. Thus, our neuroblastoma transfectants help demonstrate stress-induced regulation of the 5-HT1A-R, which in turn exerts a strong and cell type-specific control over such essential cell-surface determinants like gangliosides.

Animals↗