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V Shankar

Publications and source records attributed to V Shankar.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning of the carboxy terminus of a canine tracheobronchial mucin.

A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCTM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CTM-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCTM-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin.

Amino Acid Sequence↗
PubMed1992

Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis.

Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3'-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5'-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5'-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5'-AMP effectively, suggests the involvement of histidine in catalysis.

Aspergillus oryzae↗
PubMed1992

Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding.

Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.

Adenosine Monophosphate↗
PubMed1992

Active-site characterization of S1 nuclease. I. Affinity purification and influence of amino-group modification.

A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3'-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.

Animals↗
PubMed1992

Kinetics of virus spread and changes in levels of several cytokine mRNAs in the brain after intranasal infection of rats with Borna disease virus.

We have used the reverse transcriptase-polymerase chain reaction technique to gain insight into the pathogenesis of encephalitis caused by Borna disease virus (BDV). RNA specific for BDV was first detected in the olfactory bulb of intranasally infected rats at 6 days postinfection (p.i.). At 14 days p.i., high levels of BDV RNA were found in all brain regions, and at 26 days p.i., BDV-specific RNA was also present in the eye, nasal mucosa, and facial skin. In the chronic phase of the disease, BDV RNA was identified in many peripheral organs but not in blood. Analysis of brain tissue for the presence of cytokine mRNAs revealed that the mRNA levels of interleukin-6 (IL-6), tumor necrosis factor alpha, and IL-1 alpha had increased sharply at 14 and 26 days p.i. These cytokine mRNAs reached maximum levels at the peak of inflammatory reactions and decreased drastically in the chronic phase of the disease. Although IL-2 mRNA was also found in normal brain, it was markedly increased in BDV-infected brain at 14 days p.i. Expression of gamma interferon (IFN-gamma) mRNA, which was not observed in normal rat brain, was detected at 14 days p.i. and reached a maximum level at 38 days p.i. IL-2 and IFN-gamma mRNA expression correlated with expression of CD4 and CD8 mRNAs, indicating that both CD4+ and CD8+ T lymphocytes are induced in the early stages of BDV infection. Since IFN-gamma and CD8 mRNA levels were still highly elevated in the chronic phase of Borna disease, it is likely that CD8+ T lymphocytes act to reduce inflammation and to ameliorate neurological signs during the chronic phase of infection.

Animals↗
PubMed1992

Endothelin inhibits osteoclastic bone resorption by a direct effect on cell motility: implications for the vascular control of bone resorption.

The abundance of endothelin (ET)-producing endothelial cells in bone marrow and the proximity of these cells to bone-resorbing osteoclasts prompted us to evaluate the action of ET-1 on osteoclast function. Osteoclasts disaggregated from neonatal rat long bones were settled onto devitalized cortical bone substrate, and resorption was quantified by morphometry. The supernatant tartrate-resistant acid phosphatase activity was determined by a spectrophotometric method using paranitrophenol phosphate as substrate. Cell motility was quantified by time lapse video- and computer-assisted image processing using an empirical procedure for morphometric analysis. Cytosolic free calcium levels ([Ca2+]i) were measured in single cells by an indo 1-based microspectrofluorimetric method. Using the area of bone resorbed per slice as response, we found that ET-1 caused a significant (P = 0.011) concentration-dependent inhibition of osteoclastic bone resorption (EC50 = 2.5 nM) without inhibiting acid phosphatase secretion. Exposure of isolated osteoclasts to ET-1 also led to a marked concentration-dependent inhibition of osteoclast motility (EC50 = 7.9 nM; P = 0.013; t1/2 = 18 min) without significant effects on cell spread area. These effects of ET-1 were reversible after removing the peptide, and the cells remained viable during the experiments. In addition, ET-1 did not elevate [Ca2+]i at the concentrations tested. The results suggest that ET-1 specifically interacts with an osteoclast receptor to inhibit osteoclastic bone resorption and cell motility. As the concentration of ET-1 required for osteoclast inhibition was similar to that reported for smooth muscle contraction, it is possible that ET-1, produced locally from the bone marrow endothelial cell, might play a primary role in osteoclast regulation.

Acid Phosphatase↗
PubMed1992

Partial purification and immobilization of ribonuclease T2.

A simple procedure, consisting of water extraction, heat treatment at pH 2.0, negative adsorption on DEAE-cellulose at pH 4.9, and concanavalin A-Sepharose chromatography, was developed for the partial purification of ribonuclease (RNase) T2 from taka-diastase powder with an overall yield of 5.5%. The partially purified enzyme when coupled to aminoethyl Bio-Gel P-60, retained 12-16% of the activity of the soluble enzyme. Temperature stability studies on RNase T2 bound to matrices, activated with increasing concentrations of glutaraldehyde, and the influence of lysine modification on the activity of the soluble enzyme revealed that the low activity observed for the gel-bound enzyme is probably due to the masking of the active site of the enzyme as a result of the involvement of lysine residues, situated near the active site, during coupling. Immobilization did not affect the pH and temperature optima of RNase T2. On repeated use, the bound enzyme retained approximately 55% of its initial activity after six cycles. These results are discussed, taking into consideration the factors affecting immobilized enzymes.

Adsorption↗
PubMed1991

Macromolecular properties and polymeric structure of canine tracheal mucins.

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.

Amino Acids↗
PubMed1991

Type O renal glucosuria.

We observed a 7-year-old boy with virtual absence of renal tubular glucose reabsorption (type O renal glucosuria). Glucose titration studies in his family revealed severe type A renal glucosuria in a younger brother, a mild type A defect in the mother and normal glucose reabsorption in the father; thus a spectrum of renal glucose transport defects was observed in members of the same family.

Biological Transport↗
PubMed1991

Direct entry of rabies virus into the central nervous system without prior local replication.

Rabies virus pathogenesis was studied in a mouse model by inoculation of the masseter muscle. At different intervals, the masseter muscle, trigeminal ganglia, and brain were analyzed for virus-specific RNA with a polymerase chain reaction assay, which revealed that as early as 18 h postinfection (p.i.), virus-specific RNA was present in the trigeminal ganglia, and at 24 h p.i., viral RNA was identified in the brain stem. Analysis of the masseter muscle demonstrated virus at 1 h p.i. but no virus-specific RNA between 6 and 30 h p.i., indicating that virus invaded the nerve ending directly, without prior replication in the muscle. At 36 h p.i., viral RNA was detected again in the masseter muscle. Selective amplification of plus- and minus-strand RNA isolated from the masseter muscle at 96 h p.i. revealed that the majority of the rabies virus-specific RNA was in the positive sense, suggesting virus replication in muscle tissue during late stages of infection.

Animals↗
PubMed1991

Translation of messenger RNA from canine tracheal epithelial cells: identification of mucin core protein.

A high-molecular-weight mucin (Mr approximately 11.0 x 10(6)) was purified from canine tracheal pouch secretions. The mucin was deglycosylated by treatment with trifluoromethane sulfonic acid for 8 h at 8 degrees C and subsequently with alpha-N-acetylgalactosaminidase. These treatments almost completely removed the carbohydrate moieties. The amino acid compositions of the deglycosylated and native mucins were similar, indicating that the deglycosylation procedure used did not cause notable degradation of the protein core. Antiserum specific for deglycosylated canine tracheal mucin was produced by immunization of rabbit with the antigen. RNA was isolated from fresh canine tracheal epithelial cells by extraction with guanidine isothiocyanate/hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ RNA. The poly(A)+ RNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine and [3H]leucine as radiolabels. The translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum to deglycosylated mucin. A labeled product of molecular weight 72,000 was present in the immunoprecipitate. When canine liver poly(A)+ RNA was used as control, no radioactivity above background was detected in the immunoprecipitate. It is concluded that the primary translation product of the canine tracheal epithelial cells is a 72,000-D protein and the monomer subunit of the mucin is about 167,000 D. Thus, in the native state, the canine tracheal mucin consists of several associating subunits.

Amino Acids↗
PubMed1990

Polymeric structure of human respiratory mucin: studies on two protein components released upon reduction of disulfide bonds.

A major mucus glycoprotein (mucin) was purified from the tracheobronchial secretions of an asthmatic patient. Upon SDS-composite gel electrophoresis, the purified native (non-reduced) mucin gave a single band. SDS-gel electrophoresis on 6% polyacrylamide gels showed the absence of low molecular mass protein contaminants. However, SDS-PAGE (6% gels) of the reduced mucin showed the presence of a major high molecular mass mucin component and two low molecular mass components of 118 and 70 kDa, respectively. The 118 and 70 kDa components were purified by preparative electroelution of the reduced mucin. These components were also separated from the reduced mucin by gel-permeation chromatography on a Superose 6 column. Chemical compositional analyses showed that the 118 kDa component was a glycoprotein while the 70 kDa component was non-glycosylated. The effect of disulfide bond reduction on mucin structure and the hydrophobic probe binding properties of native and reduced mucin were studied using the fluorescent probe technique. Mansylphenylalanine was used as the fluorescent probe. The native mucin showed the presence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the probe. On the other hand, reduced-alkylated mucin containing the 118 and 70 kDa components showed the presence of additional high-affinity (KD approximately 10(-6) M) binding sites as well as low-affinity binding sites for the probe. Reduced alkylated mucin devoid of the 118 and 70 kDa components showed the presence of only low-affinity binding sites. These observations suggest that the availability of high-affinity probe binding sites upon reduction of mucin disulfide bonds may be either due to binding of the probe to the released component(s) and/or due to noncovalent interaction of the released component(s) with the mucin causing a conformational change in the mucin structure. Thus, the 118 and 70 kDa components appear to be an integral part of the total polymeric structure of the human respiratory mucin.

Adolescent↗
PubMed1990

Over-expression of the epidermal growth factor receptor in human breast cancer cells fails to induce an estrogen-independent phenotype.

An association exists in primary human breast tumors between high epidermal growth factor receptor (EGFR) expression and a reduced number or even absence of estrogen receptors (ER). To determine whether an increase in EGFR expression might alter the estrogen responsiveness of an ER-positive human breast cancer cell line, ZR 75-1 cells were cotransfected with a plasmid containing the full-length cDNA for the human EGFR under the transcriptional control of the Harvey murine sarcoma virus (HaMSV) long terminal repeat (LTR) and with a pSV2neo plasmid. Two of the isolated G418-resistant clones were found to constitutively express EGFR levels 15- to 60-fold higher than those found on nontransfected ZR 75-1 cells. The EGFR in these clones were functionally normal since EGF could increase their autophosphorylation and since EGF could enhance the transphosphorylation of p185erbB-2. No change was seen in either the number or affinity of ER in these clones. In addition, the ability of estrogen to stimulate the anchorage-dependent and anchorage-independent growth of these clones was not significantly modified. These results suggest that an increase in EGFR expression alone is not sufficient to induce a hormone-independent phenotype in vitro in human breast cancer cells.

Blotting, Northern↗
PubMed1990

Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties.

Hydrophobic binding properties of purified human respiratory mucins were studied by the fluorescence probe technique using mansylphenylalanine (Mns-Phe) as the fluorescent probe. Mucins were purified from tracheobronchial secretions of cystic fibrosis (CF) and asthmatic patients, as well as from individuals with normal lungs, according to a protocol earlier established in our laboratory. Purified mucins were subjected to reduction-alkylation and Pronase digestion to study the effects of these treatments on the hydrophobic properties of the mucins. In addition, the effects of increased NaCl concentration on the hydrophobic properties of native and reduced-alkylated mucins were also investigated. Native mucins showed evidence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the hydrophobic ligand Mns-Phe and had between 40 and 50 binding sites/mg of mucin. Reduction of mucin using dithiothreitol in the presence of 6 M guanidine hydrochloride and subsequent alkylation with iodoacetamide apparently caused marked conformational changes in the mucin molecules as revealed by the presence of both high-affinity (KD approximately 10(-6) M) and low-affinity (KD approximately 10(-5) M) binding sites for the probe and an increase in the number of probe binding sites. Pronase digestion of the native and reduced-alkylated mucins almost completely eliminated binding of the fluorescent probe to the mucins, showing that the binding sites are on the nonglycosylated, Pronase-sensitive portion of the mucin molecules. Increasing NaCl concentrations (0.03-1.0 M) did not appreciably alter the native mucin-induced Mns-Phe fluorescence, while that of the reduced-alkylated mucin-induced Mns-Phe fluorescence was progressively increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1990

Radio-active iodine uptake in vitiligo.

Vitiligo and thyroid disease are commonly associated disorders. Twenty-two clinically euthyroid vitiligo patients were studied for functional assessment of thyroid by radioactive iodine uptake assay. Half of them showed abnormal uptake values at 24 hours. Of these patients, 90% had lower values indicating a tendency towards developing hypothyroid state. Subclinical thyroid dysfunction in vitiligo appears to be an adaptive change.

Adult↗
PubMed1990

Plasma renin activity in shock due to aluminium phosphide poisoning.

Plasma renin activity (PRA) was estimated in 30 patients with aluminium phosphide (AIP) poisoning (study group) admitted in shock. Ten patients in shock other than due to AIP poisoning (Group II A) and 20 normal healthy subjects (Group II B) served as controls. The PRA was significantly higher in the study group and group II A as compared to normal healthy subjects (p less than 0.001). Significantly higher PRA was found in the study group as compared to Group II A (p less than 0.001). The initial higher PRA continued to rise further in the study group but it started decreasing in Group II A as the duration of shock advanced. Continuation of shock in AIP poisoning was probably due to slow release of toxic PH3 gas, which was detected by positive silver nitrate paper test. The rise in PRA was directly proportional to the dose of pesticide consumed. There was direct relationship of mortality with increased PRA. Angiotensin converting enzyme inhibitors may have a role in combating shock in AIp poisoning.

Adolescent↗
PubMed1989

Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA.

To determine whether the enhanced expression of transforming growth factor alpha (TGF alpha) is sufficient to induce the neoplastic transformation of an immortalized population of mammary epithelial cells, we cotransfected NOG-8 cells, a cloned mouse mammary epithelial cell line, with a simian virus 40-human TGF alpha cDNA expression vector plasmid and a pSV2neo plasmid. After cotransfection, nine G418-resistant NOG-8 colonies were cloned and expanded. All clones were subsequently analyzed for TGF alpha mRNA expression by northern blot analysis, TGF alpha secretion, anchorage-dependent growth in serum-free medium, anchorage-independent growth in soft agar, and tumorigenicity in nude mice. Three TGF alpha-transfected NOG-8 clones expressed high levels of a specific TGF alpha mRNA, secreted elevated levels of TGF alpha into the culture medium (177-595 ng/10(8) cells/48 h), exhibited an enhanced growth rate, grew aggressively as colonies in soft agar, and formed undifferentiated, invasive carcinomas in nude mice. A neutralizing mouse monoclonal antibody generated against the low molecular weight human TGF alpha peptide was able to inhibit colony formation in soft agar by TGF alpha-transfected NOG-8 clones that produced high levels by TGF alpha. This inhibition suggested that TGF alpha acted through an external autocrine loop. NOG-8 cells and NOG-8 cells transfected with a pSV2neo plasmid alone secreted very low levels of TGF alpha, failed to grow as colonies in soft agar and did not form tumors in nude mice. These results demonstrate that overexpression of a human TGF alpha cDNA in immortalized, nontransformed mouse mammary epithelial cells can induce a transformed phenotype in vitro and can facilitate tumor formation in vivo.

Animals↗