Biomedical subjects
A Kumar
Publications and source records attributed to A Kumar.
Targeting the minor groove of DNA: crystal structures of two complexes between furan derivatives of berenil and the DNA dodecamer d(CGCGAATTCGCG)2.
Crystal structures are reported of complexes of two novel furan derivatives of berenil with alkyl benzamidine groups bound to the DNA sequence d(CGCGAATTCGCG)2. They have both been determined to 2.2 A resolution and refined to R factors of 16.9% and 18.6%. In both structures the alkyl substituents, cyclopropyl and isopropyl, are found to be orientated away from the floor of the minor groove. The drugs are located in the minor groove by two strong amidinium hydrogen bonds, to the O2 of the thymines situated at the 5' and 3' ends of the AT-rich region. The isopropyl-substituted derivative has a tight hydrogen-bonded water network in the minor groove at one amidine site, which alters the orientation of the isopropyl substituent. This compound has superior DNA-binding properties and activity against Pneumocystis carinii and Cryptosporidium parvum infections in vivo compared to the cyclopropyl derivative, which in turn is superior to the parent furan compound. We suggest that the nature and extent of the interactions of these compounds in the DNA minor groove play an important role in these activities, possibly in conjunction with a DNA-binding protein. The overall effect of these alkyl benzamidine substitutions is to increase the binding of the drugs to the minor groove.
Structural organization and chromosomal mapping of JAK3 locus.
In this study, we have isolated the genomic DNA clone encoding the murine JAK3 gene and determined its sequence. Partial genomic clones of the JAK1 and JAK2 genes encoding the tyrosine kinase domain were also isolated and compared with JAK3. The genomic structure of JAK3 consists of 23 exons. The exon/intron boundaries and the distribution of coding sequences within the exons of each of the three JAK genes were found to follow a similar pattern suggesting that various members of the JAK family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. Using in situ hybridization and FISH analysis, we have mapped the JAK3 gene to human chromosome 19p13.1-p13.2.
Inhibition of HIV-1 Rev-RRE interaction by diphenylfuran derivatives.
The interactions between RNA structures, such as RRE in the HIV-1 genome, and proteins, such as Rev of HIV-1, are essential for efficient viral replication. Compounds that bind specifically to such RNAs and disrupt their protein complexes offer a novel mechanism for inhibition of replication of the virus. As a step in this approach, we have designed and characterized a series of synthetic diphenylfuran cations that selectively inhibit Rev binding to RRE. Fluorescence titrations and gel band-shift results indicate that the diphenylfurans bind to RRE and inhibit Rev complex formation in a structure-dependent manner. The derivative with the greatest affinity for RRE has an association constant of greater than 10(7) M-1 and inhibits formation of the Rev--RRE complex at concentrations below 1 microM. It binds to RRE considerably more strongly than it binds to simple RNA duplexes. Spectral changes and energy transfer results on complex formation suggest that the compound has a nonclassical intercalation binding mode. CD studies with modified RRE hairpins indicate that the active diphenylfurans bind at the structured internal loop of RRE and cause a conformational change. The most active diphenylfurans are tetracations that appear to bind to RRE by a threading intercalation mode and cause a conformational change in the RNA that is essential for inhibition of Rev complex formation with RRE.
The domains of mammalian base excision repair enzyme N-methylpurine-DNA glycosylase. Interaction, conformational change, and role in DNA binding and damage recognition.
Repair of a variety of alkylated base adducts in DNA is initiated by their removal by N-methylpurine-DNA glycosylase. The 31-kDa mouse N-methylpurine-DNA glycosylase, derived by deletion of 48 amino acid residues from the 333-residue wild type protein without loss of activity, was analyzed for the presence of protease-resistant domains with specific roles in substrate binding and catalysis. Increasing proteolysis with trypsin generated first a 29-kDa polypeptide by removal of 42 amino-terminal residues, followed by production of 8-, 6-, and 13-kDa fragments with defined, nonoverlapping boundaries. The 8- and 13-kDa domains include the amino and carboxyl termini, respectively. Based on DNA-affinity chromatography and the protease protection assay, it appears that the 6- and 13-kDa domains are necessary for nontarget DNA binding and that the 8-kDa domain, in cooperation with the other two domains, participates in recognition of damaged bases. Furthermore, chemical cross-linking studies indicated that, in the presence of substrate DNA, the 8- and 6-kDa domains undergo conformational changes reflected by both protection from proteolysis and reduced availability of cysteine residues for the thiol-exchange reaction.
K models and type IIB superstring backgrounds.
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Comparative role of phosphotyrosine kinase domains of c-ros and c-ret protooncogenes in metanephric development with respect to growth factors and matrix morphogens.
Receptor-like protooncogenes, with tyrosine kinase catalytic domains, are expressed in neoplastic and fetal tissues and potentially have a role in embryonic development. Which protooncogene may have the dominant role in embryonic renal development during the "postinductive" period, i.e., Day 10 onward, was addressed in this study by utilizing an in vitro organ culture system. The role of various receptor-like protooncogenes, with the emphasis on c-ros and c-ret, was investigated by antisense-oligodeoxynucleotide (ODN) gene-targeting strategies at a point in metanephric development when reciprocal-inductive interactions between the epithelium and mesenchyme have already been initiated and are rampant. Also, their relationship with other morphogens, like extracellular matrix (ECM) proteins and growth factors, was studied. Initial in situ hybridization and RT-PCR analyses revealed a similar spatiotemporal expression for both c-ros and c-ret in the embryonic kidneys. At Day 13, they were mainly expressed in the developing nephrons in the nephrogenic zone and ureteric bud branches, where the signals from the mesenchymal ligands are transduced to the epithelial cell surface receptors. Minimal expression was observed in the newborn kidneys. Inclusion of antisense ODNs, derived from the phosphotyrosine kinase domains, inhibited metanephric growth in the organ culture; the most dramatic effects were observed with the c-ret antisense ODN. The c-ret-induced dysmorphogenetic effects were characterized as a decrease in the population of nephrons, atrophy of the mesenchymal cells, and loss of acuteness of the tips of ureteric bud branches. Interestingly, the ureteric bud branches continue to grow in the atrophic mesenchyme. Both c-ros and c-ret antisense ODNs reduced the gene expression and biosynthesis of various ECM proteins. The proteoglycans, expressed at the epithelial:mesenchymal interface, were most adversely affected, especially by the c-ret antisense. The treatment of metanephric explants with c-ret did not affect the gene expression of c-ros and vice versa. The specificity of the effects of c-ret antisense was also reflected by a decrease of anti-Ret protein immunoreactivity. The studies were extended to establish a relationship between c-ret protooncogene and some of the growth factors which are known to influence renal development via their tyrosine kinase-like receptors localized in the ureteric bud branches, the site apparently where c-ret is also expressed. Among the various growth factors examined, transforming growth factor-alpha (TGF-alpha) and insulin like growth factor-I (IGF-I) had the most notable trophic effects on metanephric explants and caused maximal phosphorylation of Ret protein. In addition, concurrent exposure of TGF-alpha or IGF-I and c-ret antisense ODN explants caused partial recovery from the c-ret-induced dysmorphogenetic effects in the metanephroi. The data suggest that, although a number of protooncogenes share similar catalytic domains, c-ret plays a major role during the "postinductive" period of metanephric development by perturbing the growth factor-dependent expression of ECM morphogenetic macromolecules, notably that of the proteoglycans, and also by affecting certain yet undefined growth factor-mediated phosphorylation mechanism(s) involving c-ret.
Evidence concerning the effect of topology on electrical switching in chalcogenide network glasses.
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Peptides derived from the interferon-induced PKR prevent activation by HIV-1 TAR RNA.
The double-stranded RNA-dependent protein kinase (PKR) is believed to mediate cellular antiviral responses, function as a tumor suppressor, and regulate cell growth and differentiation. Its activation is dependent on double-stranded RNA (dsRNA) structures but these interactions are not fully understood. The possibility of direct interaction between dsRNA and the arginine and lysine-rich region of PKR (residues 54-74) was examined using synthetic peptides. We found that addition of a synthetic peptide corresponding to residues 54-74 of murine PKR or residues 60-80 of human PKR inhibited the autophosphorylation and activation of the kinase by either poly(I)-poly(C) or the 82-nucleotide-long TAR RNA. Gel-shift analysis indicated that the peptides disrupted the kinase-TAR complex by binding directly to TAR RNA. These findings delineate at least one dsRNA-binding domain in PKR which may be important for its cellular activation.
M theory on orientifolds of K3 x S1.
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Orientifolds and type II dual pairs.
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IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-alpha.
IL-10, an immunoregulatory cytokine produced by T cells and monocytes, inhibits the expression of inflammatory and hemopoietic cytokines as well as its own expression. To evaluate the regulation of IL-10 production by T cells and monocytes, we measured IL-10 levels by ELISA in supernatants of PHA-stimulated PBMC following depletion of either T cells or monocytes. IL-10 production was significantly down-regulated in both T cell- and monocyte-depleted PBMC compared with undepleted PBMC, and IL-10 production could be restored by the addition of monocyte-conditioned medium (supernatant of PHA-stimulated, T cell-depleted PBMC), suggesting that IL-10 production by T cells is regulated by a monokine(s) produced by activated monocytes. To further clarify the monokine(s) responsible for IL-10 induction, we stimulated monocyte-depleted PBMC, purified CD4+, and CD8+ T cells with PHA and measured IL-10 production by ELISA and semiquantitative reverse transcriptase-PCR following monokine(s) addition. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte-depleted PBMC in a dose-dependent and additive manner. Furthermore, anti-IL-6 and anti-IL-12 Abs neutralized the IL-10-inductive effect of monocyte-conditioned medium. Similarly, IL-12 and IL-6 induced IL-10 production by purified CD4+ and CD8+ T cells. With respect to regulation of IL-10 produced by monocytes, TNF-alpha was found to induce IL-10 production by resting as well as by LPS-stimulated purified monocytes/macrophages. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and/or IL-6 can induce the expression of IL-10 by PHA-stimulated T cells, whereas TNF-alpha induces IL-10 production by monocytes. Since IL-10 inhibits the production of IL-6, IL-12, and TNF-alpha, these results may indicate a potential mechanism of negative feedback regulation of the immune response.
Biochemical characterization of Alzheimer's soluble amyloid beta protein in human cerebrospinal fluid: association with high density lipoproteins.
The soluble form of Alzheimer's amyloid beta protein (sA beta) is associated with high density lipoproteins (HDL) in normal human plasma (BBRC, 1994, 205, 1164-1171). Since sA beta is also present in cerebrospinal fluid (CSF) and the lipoprotein pattern of CSF is different from that of plasma, it was of interest to ascertain whether the interaction of sA beta with HDL also occurs in CSF. Normal human CSF lipoproteins were obtained by sequential flotation ultracentrifugation and analyzed for the presence of sA beta via immunoblot, size-exclusion chromatography, immunoelectron microscopy, N-terminal sequence and mass-spectrometry analyses. Soluble A beta was associated with CSF-HDL particles of 16.8 +/- 3.2 nm in diameter and approximately 200 kDa of relative molecular mass. A approximately 4.3 kDa component purified by HPLC was immunoreactive with anti-A beta antibodies and exhibited an N-terminal sequence identical to the A beta peptide with a mass of 4325.1 Da, indicating that the main sA beta specie associated with CSF-HDL is A beta 1-40.
Identification and analysis of the human and murine putative chromatin structure regulator SUPT6H and Supt6h.
We have isolated and sequenced SUPT6H and Supt6h, the human and murine homologues of the Saccharomyces cerevisiae and Caenorhabditis elegans genes SPT6 (P using 1603 aa = 6.7 e-95) and emb-5 (P using 1603 aa = 7.0 e-288), respectively. The human and murine SPT6 homologues are virtually identical, as they share >98% identity and >99% similarity at the protein level. The derived amino acid sequences of these two genes predict a 1603-aa protein (human) and a 1726-bp protein (mouse), respectively. There were several known features, including a highly acidic 5'-region, a degenerate SH2 domain, and a leucine zipper. These features are consistent with a nuclear protein that regulates transcription, whose extreme conservation underscores the likely importance of this gene in mammalian development. Expression of human and murine SPT6 homologues was analyzed by Northern blotting, which revealed a 7. 0-kb transcript that was expressed constitutively. The SPT6 homologue was mapped to chromosome 17q11.2 in human by somatic cell hybrid analysis and in situ hybridization. These data indicate that SUPT6H and Supt6h are functionally analogous to SPT6 and emb-5 and may therefore regulate transcription through establishment or maintenance of chromatin structure.
Role of the "helix clamp" in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis.
Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the "helix clamp" (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template.primer (T.P) binding as revealed by a lack of an effect on Km, T.P, Ki for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, koff, T.P and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T.P (L282A and R284A) demonstrated that these mutant enzymes could bind T.P stoichiometrically and tightly. In contrast, active site concentrations determined from "burst" experiments suggest that the lower activity is due to a smaller populations of enzyme bound productively to T.P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3'-primer terminus, are suggested to be critical for DNA binding and fidelity.
Alpha-subunit of farnesyltransferase is phosphorylated in vivo: effect of protein phosphatase-1 on enzymatic activity.
Farnesyltransferase is a heterodimer consisting of a 49 kDa alpha-subunit and a 46 kDa beta-subunit. In this report, we demonstrate that the endogenous heterodimeric farnesyltransferase protein is phosphorylated at the alpha-subunit in vivo and phosphorylation plays a role in the regulation of farnesyltransferase activity. In vivo 32P-labeling of PC-12 cells followed by immunoprecipitation with specific anti rat alpha-subunit IgG showed a labeled alpha-subunit protein band at an expected molecular mass of 49 kDa. Treatment of PC-12 cells with protein phosphatase inhibitor, Calyculin A, resulted in a decrease in FTase activity, and phophoserine/phosphothreonine-specific protein phosphatase-1 treatment of PC-12 and GM37 cell extracts resulted in 100% and 375% increase in farnesyltransferase activity, respectively, compared to untreated extracts.
Metabolism of azathioprine.
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