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Biomedical subjects

M Kapoor

Publications and source records attributed to M Kapoor.

At least 19 recordsLinked to original sources
PubMed2002

p53 and Fas ligand are required for psoralen and UVA-induced apoptosis in mouse epidermal cells.

A combination of 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) radiation (320-400 nm) (PUVA) is widely used in the treatment of psoriasis and other skin diseases. PUVA is highly effective in eliminating hyperproliferative cells in the epidermis, but its mechanism of action has not been fully elucidated. In this study, we used immortalized JB6 mouse epidermal cells, p53(-/-), and Fas ligand deficient (gld) mice to investigate the molecular mechanism by which PUVA induces cell death. The results indicate that PUVA treatment induces apoptosis in JB6 cells. In addition, PUVA treatment of JB6 cells results in p53 stabilization, phosphorylation, and nuclear localization as well as induction of p21(Waf/Cip1) and caspase-3 activity. In vivo studies reveal that PUVA treatment induces significantly less apoptosis in the epidermis of p53(-/-) mice compared to p53(+/+) mice. Furthermore, FasL-deficient (gld) mice are completely resistant to PUVA-induced apoptosis compared to wild-type mice. These results indicate that PUVA treatment induces apoptosis in mouse epidermal cells in vitro and in vivo and that p53 and Fas/Fas ligand interactions are required for this process, at least in vivo. This implies that similar mechanisms may be involved in the elimination of psoriatic keratinocytes from human skin following PUVA therapy.

Active Transport, Cell Nucleus↗
PubMed2002

Improved polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state (SSF) fermentation.

AIMS: To investigate the effect of amino acids, vitamins and surfactants on polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state fermentation (SSF). METHODS AND RESULTS: Bacillus sp. MG-cp-2 was isolated from the outer covering of the seeds of Celastrus paniculatus. Out of the various surfactants, amino acids and vitamins, Tween-60, DL-serine and folic acid maximally enhanced polygalacturonase production by 2.7-fold (240.0 U x ml(-1)), 4.0-fold (360.0 U x ml(-1)) and 3.8-fold (342.0 U x ml(-1)) respectively, under submerged fermentation (SmF). In solid state fermentation (SSF), Tween-80, pyridoxine and DL-ornithine monohydrochloride induced highest enzyme production up to 1.73-fold (6956.5 U x g(-1)), 5.3-fold (21224.4 U x g(-1)) and 5.74-fold (23076.9 U x g(-1)), respectively. CONCLUSION: Amino acids and their analogues, vitamins and surfactants effect significantly polygalacturonase production by Bacillus sp. MG-cp-2 when grown under submerged (SmF) and solid state fermentation (SSF) conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides useful information about regulation of polygalacturonase biosynthesis in Bacillus sp. MG-cp-2, which appears to be an interplay of nutritional and physical factors. Alkaline polygalacturonase from Bacillus sp. MG-cp-2 will be extremely useful in the treatment of alkaline pectic waste waters from vegetable and fruit processing industries and in degumming of bast fibres.

Amino Acids↗
PubMed2002

Minimal promoter for the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa.

The expression of the NAD+-specific glutamate dehydrogenase (NAD-GDH) gene of Neurospora crassa is subject to catabolite repression. To identify the minimal sequence necessary for promoter function, the 5'-flanking region of the NAD-GDH gene was screened for potential protein-binding sites. Fragments of DNA, containing sequences upstream from the ATG initiation codon, were employed as probes of Southwestern blots of total cellular protein from cells grown in media promoting repression and induction of NAD-GDH. Two polypeptides interacted differentially with a promoter probe; one was present in greater abundance in repressed cells and a higher relative level of the second was witnessed in induced cells. Electrophoretic mobility shift assays with labeled promoter fragments exhibited preferential interaction with proteins in the induced cultures. The upstream sequence containing the putative protein-binding sites was fused with the coding sequence of the green fluorescent protein (GFP). The resulting plasmid was introduced into the microconidia of an albino mutant of N. crassa by electroporation. Stable integration of the plasmid and_expression of GFP in the hyphae and conidia of the transformants were demonstrated by Southern and Western blot analysis and fluorescence microscopy.

Base Sequence↗
PubMed2002

The oligomeric state, complex formation, and chaperoning activity of hsp70 and hsp80 of Neurospora crassa.

Molecular chaperones perform vital cellular functions under normal growth conditions and protect cells against stress-induced damage. The stress proteins Hsp70 and Hsp80 of Neurospora crassa were extracted from heat-shocked mycelium, purified to near homogeneity, and examined with respect to their oligomeric state, complex formation, and chaperoning properties. Their oligomeric state was assessed by dynamic light-scattering measurements, and both Hsp70 and Hsp80 were observed to form a range of soluble, high-molecular-mass protein aggregates. Direct interaction between Hsp70 and Hsp80 was studied by partial tryptic digestion and surface plasmon resonance (SPR). Hsp70 was immobilized on the sensor chip surface, and the binding of Hsp80 in solution was followed in real time. Proteolytic digestion revealed that Hsp70-Hsp80 complex formation results in conformational changes in both proteins. The data from SPR studies yielded an equilibrium dissociation constant, KD, of 8.5 x 10(-9) M. The chaperoning ability of Hsp70, Hsp80, and Hsp70-Hsp80 was monitored in vitro by the protection of citrate synthase from thermal aggregation. The binding of nucleotides modulates the oligomeric state, chaperoning function, and hetero-oligomeric complex formation of Hsp70 and Hsp80.

Citrate (si)-Synthase↗
PubMed2001

Kinetic determinants of the interaction of enoyl-ACP reductase from Plasmodium falciparum with its substrates and inhibitors.

We have recently demonstrated that Plasmodium falciparum, unlike its human host, has the type II fatty acid synthase, in which steps of fatty acid biosynthesis are catalyzed by independent enzymes. This difference could be successfully exploited in the design of drugs specifically targeted at the different enzymes of this pathway in P. falciparum, without affecting the corresponding enzymes in humans. The importance of enoyl-ACP reductase (FabI) in the fatty acid biosynthesis pathway makes it an important target in antimalarial therapy. We report here the initial characterization of Plasmodium FabI expressed in Escherichia coli. The K(m) values of the enzyme for crotonyl-CoA and NADH were derived as 165 and 33 microM, respectively. Triclosan shows competitive kinetics with respect to NADH but is uncompetitive with respect to NAD(+), which shows that the binding of triclosan to the enzyme is facilitated in the presence of NAD(+).

Acyl Coenzyme A↗
PubMed2001

The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization.

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 if 2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (DeltaC(p) and also the temperature dependence of the thermodynamic parameters, namely, DeltaG, DeltaH, and DeltaS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters DeltaH(g), DeltaS(g), and DeltaC(p) as 174.1 kcal x mol(-1), 0.512 kcal x mol(-1) x K(-1), and 3.41 kcal x mol(-1) x K(-1), respectively, at T(g) = 339.4 K. Also, the free energy of unfolding, DeltaG(s), at its temperature of maximum stability (T(s) = 293 K) is 13.13 kcal x mol(-1). Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined DeltaC(p) (3.2 +/- 0.28 kcal x mol(-1) x K(-1)) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small DeltaC(p) for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.

Agglutinins↗
PubMed2001

Bcl-2 expression in F-MuLV-induced erythroleukemias: a role for the anti-apoptotic action of Bcl-2 during tumor progression.

Erythroleukemias induced by various strains of Friend virus are multistage malignancies that result from the accumulation of genetic mutations, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In this study, we demonstrate that Bcl-2 expression is activated in the majority of F-MuLV-induced erythroleukemia cell lines. In contrast, Bcl-2 was not expressed in any of the FV-P-induced erythroleukemia cell lines and protein levels were low or negligible in FV-A-induced erythroleukemia cell lines examined. In vivo, Bcl-2 expression levels gradually increased in F-MuLV-induced erythroleukemic cells prior to adaptation to culture. High expression of Bcl-2 in F-MuLV-induced erythroleukemic cells was shown to proceed the emergence of p53 mutation suggesting that Bcl-2 expression may delay p53 mutation in the leukemic cells. This is further supported by the demonstration that the majority of F-MuLV-induced erythroleukemia cell lines established from primary tumors induced in p53 mutant mice express low to negligible levels of Bcl-2. We have shown that the high levels of Bcl-2 expression in FV-P-induced erythroleukemic cells inhibited apoptosis induced by etoposide, low serum and p53 expression. Similarly, ectopic Bcl-2 expression within these cells also provided protection from apoptosis induced by etoposide and growth in low serum. These results suggest that the anti-apoptotic action of Bcl-2 may confer a selective in vivo and in vitro growth advantage to F-MuLV-induced erythroleukemic cells, which is not shared by FV-P/FV-A-induced erythroleukemic cells. The observed induction of Bcl-2 expression in vivo constitutes a novel but late oncogenic event associated with the progression of F-MuLV-induced erythroleukemias.

Animals↗
PubMed2001

Microbial xylanases and their industrial applications: a review.

Despite an increased knowledge of microbial xylanolytic systems in the past few years, further studies are required to achieve a complete understanding of the mechanism of xylan degradation by microorganisms and their enzymes. The enzyme system used by microbes for the metabolism of xylan is the most important tool for investigating the use of the second most abundant polysaccharide (xylan) in nature. Recent studies on microbial xylanolytic systems have generally focussed on induction of enzyme production under different conditions, purification, characterization, molecular cloning and expression, and use of enzyme predominantly for pulp bleaching. Rationale approaches to achieve these goals require a detailed knowledge of the regulatory mechanism governing enzyme production. This review will focus on complex xylan structure and the microbial enzyme complex involved in its complete breakdown, studies on xylanase regulation and production and their potential industrial applications, with special reference to biobleaching.

Bacteria↗
PubMed2001

Fluid and electrolyte abnormalities.

Patients with cancer are at risk for developing a variety of fluid and electrolyte disturbances caused by the disease process or by complications from therapy. An understanding of the pathophysiology of these potential abnormalities allows the clinician to manage patients expectantly and to avoid severe metabolic disarray by correcting imbalances promptly.

Acidosis↗
PubMed2001

Malignancy and renal disease.

A variety of renal diseases and electrolyte disorders may be associated with various malignancies or with treatment of malignancy with chemotherapeutic drugs or radiation. This article reviews renal disease in cancer patients, which constitutes a major source of morbidity and mortality.

Acute Kidney Injury↗
PubMed2001

A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1.

Phosphorylation of the human p53 protein at Ser-392 has been shown to be responsive to UV but not gamma irradiation. Here we describe identification and purification of a mammalian UV-activated protein kinase complex that phosphorylates Ser-392 of p53 in vitro. This kinase complex contains casein kinase 2 (CK2) and the chromatin transcriptional elongation factor FACT (a heterodimer of hSpt16 and SSRP1). In vitro studies show that FACT alters the specificity of CK2 in the complex such that it selectively phosphorylates p53 over other substrates including casein. In addition, phosphorylation by the kinase complex enhances p53 activity. These results thus provide a potential mechanism for p53 activation by UV irradiation.

Amino Acid Sequence↗
PubMed2001

Changes in availability of oxygen accentuate differences in capsular polysaccharide expression by phenotypic variants and clinical isolates of Streptococcus pneumoniae.

Most isolates of Streptococcus pneumoniae are mixed populations of transparent (T) and opaque (O) colony phenotypes. Differences in the production of capsular polysaccharide (CPS) between O and T variants were accentuated by changes in the environmental concentration of oxygen. O variants demonstrated a 5.2- to 10.6-fold increase in amounts of CPS under anaerobic compared to atmospheric growth conditions, while CPS production remained low under all conditions for T variants. Increased amounts of CPS in O compared to T pneumococci were associated with increased expression of cps-encoded proteins. The inhibitory effect of oxygen on expression of CPS in O variants correlated with decreased tyrosine phosphorylation of CpsD, a tyrosine kinase and regulator of CPS synthesis. Modulation of CpsD expression and its activity by tyrosine phosphorylation may allow the pneumococcus to adapt to the requirements of both colonization, where decreased CPS allows for adherence, and bacteremia, where increased CPS may be required to escape from opsonic clearance. In patients with invasive infection, paired isolates from the same patient were shown to have predominantly a T colony phenotype without phosphotyrosine on CpsD when cultured from the nasopharynx, and an O phenotype that phosphorylates CpsD in response to oxygen when cultured from the blood. Differences in the availability of oxygen, therefore, may be a key factor in allowing for the selection of distinct phenotypes in these two host environments.

Bacteremia↗
PubMed2000

Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp.

A thermostable and cellulase-free xylanase has been produced from Streptomyces sp. QG-11-3 in solid substrate fermentation using wheat bran and eucalyptus kraft pulp as the prime solid substrates. The maximum xylanase yield obtained using these two substrates were 2360 U/g and 1200 U/g dry solid substrate at substrate:moisture ratios of 1:3 and 1:2.5, respectively. In immobilized cell system using polyurethane foam (PUF) and three nonwoven fabrics, namely, polyester, silk, and cotton, the xylanase yields were enhanced by 2.5-fold (203 U/ml), 1.91-fold (155 U/ml), 1.54-fold (125 U/ml), and 1.47-fold (119 U/ml), respectively, compared to the xylanase yield in liquid-batch fermentation (81 U/ml). In the biobleaching experiments, the xylanase dose of 3.5 U/g moisture free pulp exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50 degrees C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 4.5% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (%ISO) by 20% and improved the pulp properties such as tensile strength and burst factor by up to 63% and 8%, respectively.

Journal Article↗
PubMed2000

High metastatic potential in mice inheriting a targeted p53 missense mutation.

To understand the relevance of p53 missense mutations in vivo, we generated a mouse containing an arg-to-his substitution at p53 amino acid 172, which corresponds to the R175H hot-spot mutation in human tumors by homologous recombination. Inadvertently, this mouse contains the additional deletion of a G nucleotide at a splice junction that attenuates levels of mutant p53 to near wild-type levels. Mice heterozygous for the mutant allele differed from p53(+/-) mice in tumor spectrum, with a significant increase in the number of carcinomas and a slight decrease in the number of lymphomas. More importantly, the osteosarcomas and carcinomas that developed in these mutant mice frequently metastasized (69% and 40%, respectively). In contrast, metastasis is rare in osteosarcomas of p53(+/-) mice. Loss of heterozygosity studies of tumors indicated loss of heterozygosity in only 1 of 11 tumors. These data indicate clear differences between a p53 missense mutation and a null allele in tumorigenesis in vivo and suggest that the p53R172HDeltag mutant represents a gain-of-function allele.

Animals↗
PubMed2000

Cooperative phosphorylation at multiple sites is required to activate p53 in response to UV radiation.

The activity of the tumor suppressor p53 is induced in response to DNA-damaging agents such as UV and gamma radiation. Phosphorylation is one of the key regulatory steps for activating p53 function. Recent reports have shown that p53 is phosphorylated at both serines 15 and 392 in response to UV radiation. Phosphorylation at serine 15 prevents the binding of HDM2, a negative regulator of p53. Phosphorylation at serine 392 induces the DNA-binding function of p53. We examined the requirement for phosphorylation at both serines and show that both these modifications occur on the same molecule of p53. In vitro assays demonstrate that phosphorylation at either one of these sites is not sufficient to yield an active p53. Phosphorylation by DNA-PK, that modifies serines 15 and 37, inhibits HDM2 binding to p53 but does not induce the DNA-binding activity of p53. Phosphorylation at serine 392, on the other hand, stimulates the DNA-binding ability of p53 but does not make it immune to binding and inhibition by HDM2. Thus, our results demonstrate that multiple sites need to be modified to yield a functional p53.

Casein Kinase II↗
PubMed2000

Tyrosinase-related protein 2 as a mediator of melanoma specific resistance to cis-diamminedichloroplatinum(II): therapeutic implications.

A major obstacle in the systemic treatment of advanced malignant melanoma is its intrinsic resistance to conventionally used chemotherapeutic agents. In order to investigate the mechanisms of this intrinsic resistance, we have previously utilized retroviral insertional mutagenesis on an early-stage, drug sensitive human melanoma cell line (WM35) to establish mutated cell lines that exhibited increased resistance to cis-diammi-nedichloroplatinum(II) (CDDP). Here, we demonstrate that this increased resistance to CDDP is mediated by the over-expression of tyrosinase-related protein-2 (TYRP2), an enzyme that normally functions in the biosynthesis of the pigment, melanin. Northern and Western blot analyses revealed that the expression of TYRP2 in the virally-derived cell lines as well as in a panel of human melanoma cell lines positively correlated with their levels of resistance to CDDP. Furthermore, enforced expression of TYRP2 in WM35 cells by transfection elevated their resistance to CDDP. The increased CDDP resistance in the virally-derived clones and TYRP2 transfectants was accompanied by a reduction in CDDP-induced apoptosis. Interestingly, the virally-derived CDDP-resistant clones also showed cross resistance to carboplatin and methotrexate, but not taxol, suggesting that TYRP2 over-expression may confer resistance specifically to DNA damaging agents. Overall, these results demonstrate a novel mechanism of drug resistance in human melanoma cells that is mediated by the over-expression of TYRP2. Since TYRP2 is expressed only in cells of melanocytic lineage, this may represent the first report of a lineage-specific mechanism of drug resistance. In summary, these findings suggest a significant role for TYRP2 in the intrinsic drug resistance phenotype of human melanoma cells and may have important implications in the development of chemosensitization strategies for the clinical management of this disease.

Antineoplastic Agents↗
PubMed2000

The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein-mediated killing.

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C-reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP-mediated serum bactericidal activity regardless of the genetic background. Differences in CRP-mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.

Alleles↗