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N Srinivasan

Publications and source records attributed to N Srinivasan.

At least 91 records · Page 5Linked to original sources
PubMed1998

Interaction of estradiol, progesterone and corticosterone on uterine connective tissue degrading enzymes.

The impact of ovarian hormones and corticosterone acetate on uterine connective tissue degrading enzymes were studied in mature albino rats. Ovariectomy resulted in a significant increase in the activities of alpha- and beta-galactosidases and glucosidases in the uterus. Administration of estradiol to ovariectomized rats brought back the activities of alpha-galactosidase and alpha-glucosidase to normalcy. While beta-galactosidase and beta-glucosidase were significantly decreased. Administration of progesterone to ovariectomized rats resulted in the increase of alpha- and beta-galactosidases and glucosidases. Administration of corticosterone to ovariectomized rats produced a further increase in alpha- and beta-galactosidases and glucosidases in the uterus. Adrenalectomy in ovary intact rats produced a decrease in alpha-galactosidase however, beta-glucosidase was significantly increased. Administration of corticosterone to ovary intact rats significantly increased the activities of alpha- and beta-galactosidases, while alpha- and beta-glucosidases were found to be decreased. Ovariectomy resulted in a significant increase in the activities of cathepsin-D and cathepsin-E. Administration of estradiol to ovariectomized rats brought back the activity of cathepsin-D to normalcy, whereas cathepsin-E was significantly increased. Administration of progesterone as well as estradiol to ovariectomized rats significantly increased the levels of cathepsin-E, however, cathepsin-D was brought back to normalcy. Administration of corticosterone to ovariectomized rats as well as ovariectomy + adrenalectomy significantly increased the activity of cathepsin-D and cathepsin-E. Adrenalectomy significantly decreased the activity of cathepsin-D, while administration of corticosterone increased the cathepsin-D and cathepsin-E in the uterus. Therefore, these results suggest that estradiol is a potent ovarian steroid protecting the extra cellular matrix components. The effect of progesterone appears to modulate and act hand in hand with estradiol. Corticosterone appears to have an opposite effect to that of estradiol.

Adrenalectomy↗
PubMed1997

Effects of inhibitors of signal transduction pathways on transforming growth factor beta1 and osteogenic protein-1-induced insulinlike growth factor binding protein-3 expression in human bone cells.

Signal transduction initiated by TGFbeta1 and OP-1 was studied in MG63 human osteosarcoma cells and in normal human bone cells (HBCs) in the presence of inhibitors of signal transduction events, using insulinlike growth factor binding protein-3 (IGFBP-3) production as an end point. Treatment of serum-free MG63 cells and normal HBCs with TGFbeta1 increased IGFBP-3 protein level several fold in the conditioned medium. This effect of TGFbeta1 was mediated by increased de novo synthesis because mRNA level increased to the same extent as protein level and TGFbeta1 treatment had very little effect on IGFBP-3 protease activity. The stimulatory effect of TGFbeta1 on IGFBP-3 production was inhibited in a dose-dependent manner by pretreatment with staurosporine, a protein kinase C inhibitor, or with vanadate, a phosphotyrosyl protein phosphatase inhibitor in both MG63 cells and normal HBCs. In addition, pretreatment with okadoic acid, an inhibitor of serine/threonine protein phosphatase, counteracted TGFbeta1 induction of IGFBP-3 production. Interestingly, pretreatment of MG63 cells or HBCs with staurosporine, vanadate, or okadoic acid augmented OP-1 stimulation of IGFBP3 production. Staurosporine- or vanadate-induced changes in IGFBP-3 protein levels in the presence of TGFbeta1 and OP-1 were associated with corresponding changes in IGFBP-3 mRNA levels in MG63 cells. These findings are consistent with the hypothesis that TGFbeta1 and OP-1 increase IGFBP-3 expression via distinct intracellular signal transduction pathways.

Bone Morphogenetic Protein 7↗
PubMed1997

Domain structure of hepatocyte growth factor/scatter factor (HGF/SF).

The modular structure of hepatocyte growth factor/scatter factor (HGF/SF) has facilitated structure-function analysis. Domain deletion experiments have established that the N-domain, kringle 1 and kringle 2 are essential for HGF/SF activity on target cells and that, conversely, truncated variants containing the N-domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) exhibit partial agonistic or antagonistic activity depending on target cells and the presence of full length HGF/SF. The 3D structures of the six domains of HGF/SF have been modelled on the structure of homologues, offering interesting insights into putative mechanisms of domain interactions, receptor binding and activation. The predictions offered by such models are currently assessed by protein engineering techniques and will ultimately be measured against experimental structures.

Gene Deletion↗
PubMed1997

Prediction of the stability of protein mutants based on structural environment-dependent amino acid substitution and propensity tables.

An approach to the prediction of mutant stability is described using knowledge of amino acid replacements that are tolerated within the families of homologous proteins of known 3-D structure. Amino acid variations in families of homologous proteins are converted to propensity and substitution tables; these provide quantitative information about the existence of an amino acid in a structural environment and the probability of replacement by any other amino acid. The tables are used to calculate a 'stability difference score', analogous to the difference in free energy between a mutant and the wild type. The method has been developed and tested using the high-resolution structures for T4 lysozyme and 159 site-specific mutants. We show that differences in stability scores are correlated with experimentally observed free energy differences and differences in melting temperature. Blind tests, using only structural information derived from the parent wild-type crystal structures, on a combined set of 83 staphylococcal nuclease and 68 barnase mutants showed a correlation of 0.80 in the predicted stability changes with experimental thermodynamic data. Approximately 86% of the predictions were correctly classified as destabilizing or stabilizing.

Algorithms↗
PubMed1997

Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementing cosmids identified four open reading frames, three of them homologous to Pseudomonas putida, E. coli, and Haemophilus influenzae exbB, exbD, and tonB genes. The N. meningitidis TonB protein is 28.8 to 33.6% identical to other gram-negative TonB proteins, while the N. meningitidis ExbD protein shares between 23.3 and 34.3% identical amino acids with other ExbD and TolR proteins. The N. meningitidis ExbB protein was 24.7 to 36.1% homologous with other gram-negative ExbB and TolQ proteins. Complementation studies indicated that the neisserial Ton system cannot interact with the E. coli FhuA TonB-dependent outer membrane receptor. The N. meningitidis tonB mutant was unable to use Hb, Hb-haptoglobin complexes, transferrin, and lactoferrin as iron sources. Insertion of an antibiotic cassette in the 3' end of the exbD gene produced a leaky phenotype. Efficient usage of heme by N. meningitidis tonB and exbD mutants suggests the existence of a Ton-independent heme utilization mechanism. E. coli complementation studies and the analysis of N. meningitidis hmbR and hpu mutants suggested the existence of another Hb utilization mechanism in this organism.

Amino Acid Sequence↗
PubMed1996

Symmetry, stability, and dynamics of multidomain and multicomponent protein systems.

Symmetry is commonly observed in many biological systems. Here we discuss representative examples of the role of symmetry in structural molecular biology. Point group symmetries are observed in many protein oligomers whose three-dimensional atomic structures have been elucidated by x-ray crystallography. Approximate symmetry also occurs in multidomain proteins. Symmetry often confers stability on the molecular system and results in economical usage of basic components to build the macromolecular structure. Symmetry is also associated with cooperativity. Mild perturbation from perfect symmetry may be essential in some systems for dynamic functions.

Protein Conformation↗
PubMed1996

Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites.

Pentraxins, which include C reactive protein (CRP) and serum amyloid P component (SAP), are prototypic acute phase reactants that serve as indicators of inflammatory reactions. Here we report genomic and cDNA cloning of mouse ptx3 (mptx3), a member of the pentraxin gene family and characterize its extrahepatic expression in vitro and in vivo. mptx3 is organized into three exons on chromosome 3: the first (43 aa) and second exon (175 aa) code for the signal peptide and for a protein portion with no high similarity to known sequences the third (203 aa) for a domain related to classical pentraxins, which contains the "pentraxin family signature." Analysis of the N terminal portion predicts a predominantly alpha helical structure, while the pentraxin domain of ptx3 is accommodated comfortably in the tertiary structure fold of SAP. Normal and transformed fibroblasts, undifferentiated and differentiated myoblasts, normal endothelial cells, and mononuclear phagocytes express mptx3 mRNA and release the protein in vitro on exposure to interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)alpha. mptx3 was induced by bacterial lipopolysaccharide in vivo in a variety of organs and, most strongly, in the vascular endothelium of skeletal muscle and heart. Thus, mptx3 shows a distinct pattern of in vivo expression indicative of a significant role in cardiovascular and inflammatory pathology.

3T3 Cells↗
PubMed1996

Structural aspects of the functional modules in human protein kinase-C alpha deduced from comparative analyses.

Three-dimensional models of the five functional modules in human protein kinase C alpha (PKC alpha) have been generated on the basis of known related structures. The catalytic region at the C-terminus of the sequence and the N-terminal auto-inhibitory pseudo-substrate have been modeled using the crystal structure complex of cAMP-dependent protein kinase (cAPK) and PKI peptide. While the N-terminal helix of the catalytic region of PKC alpha is predicted to be in a different location compared with cAPK, the C-terminal extension is modeled like that in the cAPK. The predicted permissive phosphorylation site of PKC alpha, Thr 497, is found to be entirely consistent with the mutagenesis studies. Basic Lys and Arg residues in the pseudo-substrate make several specific interactions with acidic residues in the catalytic region and may interact with the permissive phosphorylation site. Models of the two zinc-binding modules of PKC alpha are based on nuclear magnetic resonance and crystal structures of such modules in other PKC isoforms while the calcium phospholipid binding module (C2) is based on the crystal structure of a repeating unit in synaptotagmin I. Phorbol ester binding regions in zinc-binding modules and the calcium binding region in the C2 domain are similar to those in the basis structures. A hypothetical model of the relative positions of all five modules has the putative lipid binding ends of the C2 and the two zinc-binding domains pointing in the same direction and may serve as a basis for further experiments.

Amino Acid Sequence↗
PubMed1996

Motifs and conformational analysis of amino acid residues adjoining beta-turns in proteins.

Using a data set of 250 non-homologous high-resolution globular proteins, a systematic analysis of the conformations that precede and succeed (positions i and i + 3) the various classical beta-turn types has been carried out. The collective conformation of a specific beta-turn type, including the flanking positions, termed motif, has been studied. In all the four turn types, the majority of examples are preceded and succeeded by extended conformation. Some of the other observations are: (1) In a type I beta-turn, Gly at position i + 3 has a higher favorability to occur with positive phi and does not prefer the major motif beta-alpha R-alpha R-beta. (2) The left-handed alpha-helical conformation (alpha L) is not preferred at both the flanking positions for type I' and II' beta-turns. (3) The beta--beta motif is favourable for all the turn types and the motif beta--alpha L very highly favourable for type I.

Amino Acids↗
PubMed1996

Insulin-like growth factor-binding protein-6 produced by human PC-3 prostate cancer cells: isolation, characterization and its biological action.

The PC-3 human prostatic carcinoma cell line has been extensively used as a model for studies on the regulation of prostate tumor cell proliferation. Because of the importance of IGF-binding proteins (IGFBPs) in the control of IGF activities that regulate cell proliferation in normal and malignant cell types, we undertook studies to characterize the IGFBPs produced by PC-3 prostate tumor cells in culture. We previously found, using an IGF-I affinity column for purification and a polyethylene glycol (PEG) precipitation assay for IGFBP detection, that PC-3 cells in culture produced a single predominant IGFBP, IGFBP-4, which inhibits IGF activities. We now present evidence that PC-3 cells also produce IGFBP-6 in abundant quantity; in the previous study this was apparently not detected in the IGF-I-bound fraction with the PEG precipitation and Western ligand blot assays. In the current study, IGF-II affinity purification of IGFBPs produced by PC-3 cells, followed by C8 HPLC reverse-phase chromatography using a shallow acetonitrile gradient, produced two major protein peaks. N-terminal amino acid sequence of peak 1 was identical to that of IGFBP-6 while that of peak 2 was identical to that of IGFBP-4. Characterization of purified IGFBP-6 from PC-3 cells revealed properties which are distinct from other IGFBPs. PEG did not precipitate the complex of 125I-IGF-II/IGFBP-6 while it precipitated the complexes between 125I-IGF-II and other IGFBPs. Indeed, IGFBP-6 decreased the amount of 125I-IGF-II tracer in the PEG precipitate in a dose-dependent manner. Also, the binding of IGFBP-6 with 125I-IGF-II was poor in Western ligand blots compared with other IGFBPs. In studies on IGFBP-6 actions, IGFBP-6 completely inhibited IGF-II-induced [3H]thymidine incorporation in MC3T3-E1 mouse osteoblast cells while it had only minimal inhibitory effects on IGF-I-induced [3H]thymidine incorporation. This differential effect is associated with the fact that IGFBP-6 has greater affinity for IGF-II than IGF-I. The results of this study indicated that (1) Western ligand blotting is not sensitive for identification of IGFBP-6, (2) the unique behavior of IGFBP-6 in the PEG assay system necessitates the use of charcoal adsorption procedure for IGFBP-6 activity detection and (3) PC-3 cells should provide a useful model system for studying regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF actions.

Blotting, Western↗
PubMed1995

Inhibition of staphylococcal enterotoxin-driven lymphocyte proliferation by anti-MHC class II monoclonal antibody.

Staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules and the V beta region of T cell receptors (TCR) and subsequently induces T cell proliferation. This mitogenicity is the basis of pathological effects seen in food poisoning and toxic shock syndrome. Toxin-specific monoclonal antibodies have previously been shown to be effective in blocking toxin stimulated T cell responses. In this study, a monoclonal antibody, 52BL1, was found to be a potent inhibitor of SEA-, SEB-, SEC1-, SED-, and SEE-induced lymphocyte proliferation assays, which indicates that a single anti-HLA (human leukocyte antigen) class II antibody is effective in blocking the biological effects of these toxins. These results demonstrate the possibility of using anti-HLA class II antibodies in a clinical setting as an antagonist to staphylococcal enterotoxinmediated pathogenesis.

Antibodies, Monoclonal↗
PubMed1995

Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids.

Insulin-like growth factor binding proteins (IGFBPs) serve as transport proteins for the IGFs and modulate their biological effects. In order to evaluate the relative contribution of IGFBP-5 to the IGF binding capacity of serum and other biological fluids and to study more accurately the in vivo regulation of serum IGFBP-5, we have developed an RIA for IGFBP-5 using recombinant human IGFBP-5 as the antigen, tracer, and standard. The validity of the IGFBP-5 RIA was established from the following data: 1) recombinant human IGFBP-5 and purified human bone-derived IGFBP-5 inhibited the binding of tracer to the antisera in an identical manner; 2) the immunoreactive IGFBP-5 in serum co-migrated with recombinant human IGFBP-5 in both sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance gel filtration chromatography; 3) none of the other known IGFBPs (IGFBP-1, -2, -3, -4, and -6) exhibited significant cross-reactivity; 4) the addition of IGF-I or IGF-II to the samples did not affect the recovery of IGFBP-5; and 5) the recovery of the exogenously added IGFBP-5 to serum was greater than 90%. In order to further validate our IGFBP-5 RIA, measurements of IGFBP-5 were carried out in the conditioned media samples collected from human bone cells treated with effectors that are known to influence IGFBP-5 production. Treatment of human bone cells with bone morphogenetic protein-7, which increases IGFBP-5 production, caused an increase in IGFBP-5 protein levels; dexamethasone, which decreases IGFBP-5 production, caused a decrease in IGFBP-5 protein levels measured in the conditioned medium by this RIA. Application of this RIA to the measurement of the IGFBP-5 level in human serum revealed that the circulating level of IGFBP-5 in healthy women decreased from 664 +/- 108 ug/L in prepubertal girls to 222 +/- 42 ug/L in women ages 61-85 yr (P < 0.001). There was also a small but significant decrease in the mean serum level of IGFBP-5 in women ages 23-39 yr compared with the level in prepubertal girls (589 +/- 66 vs. 664 +/- 108 ug/L, P < 0.05). Additional studies with this assay may provide an insight into the physiological mechanism that regulates IGFBP-5 production in vivo and the potential role of this protein as an IGF transport protein in serum and other biological fluids.

Adolescent↗
PubMed1994

Comparative analyses of pentraxins: implications for protomer assembly and ligand binding.

BACKGROUND: Pentraxins are a family of plasma proteins characterized by their pentameric assembly and calcium-dependent ligand binding. The recent determination of the crystal structure for a member of this family, human serum amyloid P component (SAP), provides a basis for the comparative analysis of the pentraxin family. RESULTS: We have compared the sequences, tertiary structures and quaternary arrangements of SAP with human C-reactive protein (CRP), Syrian hamster SAP (HSAP) and Limulus polyphemus CRP (LIM). These proteins can adopt a beta-jelly roll topology and a hydrophobic core similar to that seen in SAP. Only minor differences are observed in the positions of residues involved in coordinating calcium ions. CONCLUSIONS: Calcium-mediated ligand binding by CRP, HSAP and LIM is similar to that defined by the crystal structure of SAP, but sequence differences in the hydrophobic pocket explain the differential ligand specificities exhibited by the homologous proteins. Differences elsewhere, including insertions and deletions, account for the different (hexameric) quaternary structure of LIM.

Amino Acid Sequence↗
PubMed1994

The recognition of protein structure and function from sequence: adding value to genome data.

The explosion of DNA sequence data from genome projects presents many challenges. For instance, we must extend our current knowledge of protein structure and function so that it can be applied to these new sequences. The derivation of rules for the relationships between sequence and structure allow us to recognize a common fold by the use of tertiary templates. New techniques enable us to begin to meet the challenge of rule-based modelling of distantly related proteins. This paper describes an integrated and knowledge-based approach to the prediction of protein structure and function which can maximize the value of sequence information.

Amino Acid Sequence↗
PubMed1994

Structure of pentameric human serum amyloid P component.

The three-dimensional structure of pentameric human serum amyloid P component at high resolution, the first reported for a pentraxin, reveals that the tertiary fold is remarkably similar to that of the legume lectins. Carboxylate and phosphate compounds bind directly to two calcium ions; interactions with a carboxyethylidene ring are mediated by Asn 59 and Gln 148 ligands of the calcium ions. These X-ray results indicate the probable modes of binding of the biologically important ligands, DNA and amyloid fibrils.

Amino Acid Sequence↗
PubMed1994

Three-dimensional model and quaternary structure of the human eye lens protein gamma S-crystallin based on beta- and gamma-crystallin X-ray coordinates and ultracentrifugation.

A 3-dimensional model of the human eye lens protein gamma S-crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3-dimensional structure of gamma-crystallin and beta-crystallin. The model is biased toward the monomeric gamma B-crystallin, which is more similar in sequence. Bovine gamma S-crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric gamma-crystallins. Because the linker has 1 extra residue compared with gamma B and beta B2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. gamma S-crystallin differs from gamma B-crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both beta- and gamma-crystallins, and may contribute to the different roles of these proteins in the lens.

Amino Acid Sequence↗