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Biomedical subjects

S Bandyopadhyay

Publications and source records attributed to S Bandyopadhyay.

At least 19 recordsLinked to original sources
PubMed2001

Successful therapy of lethal murine visceral leishmaniasis with cystatin involves up-regulation of nitric oxide and a favorable T cell response.

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4(+) T cell differentiation. A 6-fold increase in NO production was observed with 0.5 microM chicken cystatin, a natural cysteine protease inhibitor, in IFN-gamma-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-gamma. In contrast to the case with promastigotes, cystatin and IFN-gamma inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-gamma treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4(+) differentiation from Th2 to Th1, indicative of long-term resistance.

Animals↗
PubMed2000

Activation of the tumor metastasis suppressor gene, KAI1, by etoposide is mediated by p53 and c-Jun genes.

KAI1 is a metastasis suppressor gene which is capable of inhibiting the processes of tumor metastasis without affecting tumorigenicity per se. We found that etoposide, a topoisomerase II inhibitor, is able to activate the expression of the KAI1 gene in a dose-dependent manner in human prostate cancer cell lines, ALVA, DU145, and PC-3 as well as in human lung carcinoma cell A549. The activation of the KAI1 gene was mainly mediated by the c-Jun gene in the PC-3 and DU145 cell lines, while it was mediated by both p53 and c-Jun genes in the A549 cell line. These results suggest that the augmentation of the KAI1 gene expression is independently controlled by p53 and c-Jun at the transcriptional level in the human cancer cell lines. Furthermore, treatment of these cell lines with etoposide resulted in significant reduction of cellular invasion measured by the Matrigel invasion chamber. Because etoposide has been shown to be effective on advanced prostate cancer when used in combination with other regimens, our results provide further rationale to use this drug as an antimetastatic agent.

5' Untranslated Regions↗
PubMed2000

An initiator element mediates autologous downregulation of the human type A gamma -aminobutyric acid receptor beta 1 subunit gene.

The regulated expression of type A gamma-aminobutyric acid receptor (GABA(A)R) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABA(A)R subunit gene expression, including decreased beta1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABA(A)R number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABA(A)R beta1 subunit gene that is neuron specific and autologously down-regulated by GABA. beta1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABA(A)R activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3' flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of beta1 subunit mRNAs involves the differential binding of a sequence-specific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of beta1 subunit GABA(A)R gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.

Animals↗
PubMed2000

DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor.

Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1-O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3. Upon titration of lambda-repressor with single-operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions. Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, O(R)2-O(R)3-bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact. We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence.

Acrylamide↗
PubMed2000

Localization of a novel tumor metastasis suppressor region on the short arm of human chromosome 2.

Much of the lethality of malignant neoplasms is attributable directly to their ability to develop secondary growths in organs at a distance from the primary tumor mass, whereas few patients die from their primary neoplasm. Little is known about the molecular mechanism of tumor metastasis, however, which is controlled by a variety of positive and negative factors. In the search for metastasis suppressor genes, we have used the microcell-mediated chromosome transfer method and a rat prostate tumor model in SCID mice. When human chromosome 2 was introduced into the highly metastatic rat prostatic tumor cell, AT6.1, the metastatic ability of this cell was significantly (>99%) decreased in animals. An STS-based PCR analysis for 8 hybrid clones indicates that the suppressor activity is located in the p25-22 region of the chromosome. Furthermore, the AT6.1 cell with human chromosome 2 showed a reduced ability to invade Matrigel, suggesting that the suppressor activity is involved in the step of tumor invasion during the progression of prostate cancer. We have also examined the status of the suppressor region on chromosome 2 in human prostate cancer specimens and found that this region was often lost in high-grade tumors. These results suggest that the putative suppressor gene on chromosome 2 is functionally involved in the progression of human prostate cancer. Genes Chromosomes Cancer 28:285-293, 2000.

Aged↗
PubMed2000

Modulation of CD11C+ splenic dendritic cell functions in murine visceral leishmaniasis: correlation with parasite replication in the spleen.

BALB/c mice resolve Leishmania donovani infection in the liver over an 8-12-week period. However, after an initial phase of 2-4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post-infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-specific antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was purified from 30-day post-infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-gamma (IFN-gamma) production by Leishmania antigen-primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-gamma by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.

Animals↗
PubMed2000

A minimal promoter for the GABA(A) receptor alpha6-subunit gene controls tissue specificity.

The ability of nerve cells to regulate the expression of specific neurotransmitter receptors is of central importance to nervous system function, but little is known about the DNA elements that mediate neuron specific gene expression. The type A gamma-aminobutyric acid (GABA(A)) receptor alpha6-subunit gene, which is expressed exclusively in cerebellar granule cells, presents a unique opportunity to study the cis elements involved in restricting gene expression to a distinct neuronal population. In an effort to identify the regulatory elements that govern cerebellar granule cell-specific gene expression, the proximal 5' flanking regions for the human, rat, and mouse alpha6 genes were cloned and sequenced, and a major transcriptional initiation site was identified in the rodent genes. Functional analysis of rat alpha6 gene-reporter constructs in primary neuronal cultures reveals that a 155-bp TATA-less promoter region (-130 to +25 bp) constitutes a minimal promoter that can drive cerebellar granule cell-specific expression. Internal deletion and decoy competition studies demonstrate that the minimal promoter contains a 60-bp region (-130 to -70 bp) that is critical for enhanced promoter activity in cerebellar granule cells. Activity of the compromised promoter containing the deletion cannot be rescued by placing the 60-bp region downstream of the reporter gene, demonstrating that it is not a classical enhancer but rather a positionally dependent regulator. An additional cerebellar-specific activating sequence is located between -324 and -130 bp, and a downstream negative regulatory region (+158 to +294) has been shown to be active in fibroblasts but inactive in cerebellar granule cells. Taken together, the results suggest a possible mechanism for the control of cerebellar granule cell-specific expression of the GABA(A) receptor alpha6 subunit gene.

Animals↗
PubMed2000

Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis.

BACKGROUND: Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. MATERIALS AND METHODS: Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apoptosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V. RESULTS: Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-II-mediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 microM and 45.5 microM, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. CONCLUSION: Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design.

Animals↗
PubMed2000

Surveillance for polio eradication: current status and lessons learnt--India, 1999.

With the launch of the Universal Immunisation Programme in India in 1985, childhood immunisation was provided to children in all districts of the country in a phased manner by 1990. Surveillance for vaccine preventable diseases (VPD) including polio was started at the same time with monthly reporting from the districts to the Ministry of Health and Family Welfare (MOHFW), Government of India (GOI). In 1995, the Pulse Polio Immunisation (PPI) campaign was launched with the objective of polio eradication. Prior to 1997, surveillance for polio was directed at finding clinical polio cases by passive reporting from health facilities. There was no active surveillance for all cases of acute flaccid paralysis (AFP). In 1996, a scheme for the surveillance of AFP was drawn up. With the support of the Danish and US governments and Rotary International, 59 surveillance medical officers (SMOs) were hired, trained, and posted throughout the country in October 1997 to establish active surveillance of AFP. The number of SMOs was increased to 108 in August 1999. The SMOs along with their government counterparts established 10,069 reporting units nationwide by the end of November 1999 reporting weekly the occurrence of AFP cases to the district, state, and national levels; timely case investigation and collection of stool specimens from AFP cases; linkages to support the polio laboratory network; and extensive training of government counterparts. Data reported to the national level is analysed and put on an internet website which is updated every two weeks. Annualised rates of reported non-polio AFP have increased from 0.22 per 100,000 children aged < 15 years in 1997 to 1.57 in 1999. The number of polio cases associated with isolation of wild poliovirus decreased from 1404 in the third trimester of 1998 to 664 in the third trimester of 1999, yet widespread transmission of wild polioviruses persists throughout the country.

Adolescent↗
PubMed1999

DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR.

The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.

Amino Acid Sequence↗
PubMed1999

Endothelial cells express a novel, tumor necrosis factor-alpha-regulated variant of HOXA9.

The expression of the class 1 homeobox (HOX) family of "master control" transcription factors has been studied principally in embryogenesis and neoplasia in which HOX genes play a critical role in cell proliferation, migration, and differentiation. We wished to test whether HOX family members were also involved in a differentiation-like process occurring in normal, diploid adult cells, that is, cytokine-induced activation of endothelial cells (EC). Screening of a human EC cDNA library yielded several members of the A and B groups of HOX transcription factors. One clone represented a novel, alternatively spliced variant of the human HOXA9 gene containing a new exon and the expression of which was driven by a novel promoter. This variant termed HOXA9EC appeared restricted to cells of endothelial lineage, i.e. expressed by human EC from multiple sources, but not by fibroblasts, smooth muscle cells, or several transformed cell lines. HOXA9EC mRNA was rapidly down-regulated in EC in response to tumor necrosis factor-alpha due to an apparent reduction in transcriptional rate. Reporter construct studies showed that the 400 base pairs of genomic DNA directly 5' to the transcription initiation site of HOXA9EC contained the information required for both up-regulation in response to cotransfection with a HOXA9EC expression vector and tumor necrosis factor-alpha-dependent down-regulation of this gene. These results provide evidence of a novel HOX family member that may participate in either the suppression or the genesis of EC activation.

Adult↗
PubMed1999

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase.

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.

2',5'-Oligoadenylate Synthetase↗
PubMed1999

Partial anodontia in a case of Waardenburg's syndrome.

On routine examination of a case of Waardenburg's syndrome apart from the usual features of the disorder, partial anodontia involving both the lower lateral incisors was evident. The interesting association of partial anodontia and Waardenburg's syndrome is discussed along with relevant available literature.

Adolescent↗
PubMed1999

Leishmania donovani parasites interact with gamma/delta+ human peripheral blood T cells and induce susceptibility to NK cell-mediated lysis.

We recently reported that Leishmania donovani infect the human T-cell line in vitro. To examine whether primary human T cells could be infected by this parasite, a direct interaction of the peripheral blood T cells with L. donovani was examined. The percentage of gamma/delta+ T cells was markedly increased when in vitro generated normal human T-cell blasts were cultured with L. donovani amastigotes. About 30% of the gamma/delta+ T cells in the parasite exposed T-cell blasts expressed parasite antigens intracellularly without detectable intracellular parasites. Parasite exposed T-cell blasts had a reduced surface expression of HLA-DR and were lysed by the sorted CD56+ cells. In contrast, neither L. donovani amastigotes nor T-cell blasts exposed to heat killed amastigotes and/or were sensitive to the NK cell-mediated lysis. Of interest is that about 10% CD3+ peripheral blood T cells in two out of three Indian Kala-azar patients tested expressed intracellular L. donovani antigens.

Animals↗
PubMed1999

Indolylquinoline derivatives are cytotoxic to Leishmania donovani promastigotes and amastigotes in vitro and are effective in treating murine visceral leishmaniasis.

A wide variety of biologically active compounds contain indole and quinoline nuclei. Some novel indolylquinoline derivatives were synthesized from indole by Friedel-Crafts acylation reaction. Out of the four derivatives tested, 2-(2''-acetamidobenzyl)-3-(3'-indolylquinoline) (C) had no effect on the promastigotes or amastigotes of Leishmania donovani in vitro. The remaining three analogues, 2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A), 2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), and 2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D), inhibited the growth of L. donovani promastigotes in vitro and were cytotoxic to both the promastigote and amastigote forms of the parasite. These three derivatives were also effective in eliminating L. donovani amastigotes from BALB/c mouse peritoneal macrophages in vitro. One indolylquinoline derivative [A] was used to treat established visceral leishmaniasis in BALB/c mice. This compound was significantly more effective than sodium antimony gluconate (SAG) in reducing the splenic parasite load at a much lower concentration (5% of SAG). Our results suggest that indolylquinoline derivatives may be exploited as antileishmanial agents.

Animals↗
PubMed1999

Major beta-globin gene mutations in eastern India and their associated haplotypes.

324 alleles of the beta-globin gene from unrelated thalassaemia patients native to the eastern region of India (mainly from the state of West Bengal) were analysed for beta-globin gene mutations by the amplification refractory mutation system (ARMS). The major mutations that were detected are IVS-1 pos 5 (G-C), codon 26 (G-A) and codon 30 (G-C) with frequencies of 0.45, 0.33 and 0.05, respectively. Haplotype analysis revealed a very strong linkage disequilibrium of IVS-1 pos 5 (G-C) with one particular haplotype. HbE was found to be associated with two major haplotypes. Codon 30 (G-C) was associated with a haplotype that is the same as that found in the African population. Haplotype associated with codon 8/9 (+G) was the same as that found in northwest India. These findings have implications for the use of molecular diagnosis for genetic counselling and prenatal diagnosis of beta-thalassaemia in this region.

DNA Mutational Analysis↗
PubMed1999

Acute spinal cord infarction: vascular steal in arteriovenous malformation.

Central nervous system arteriovenous malformations typically present with chronic neurologic impairment. An 8-year-old boy presented with acute spinal cord infarction associated with a spinal arteriovenous malformation. Vascular steal phenomenon suggested by spinal angiography happens to underly the pathogenic mechanism.

Acute Disease↗