Search PubMed⌕ Search

Biomedical subjects

J Srinivasan

Publications and source records attributed to J Srinivasan.

At least 55 records · Page 3Linked to original sources
PubMed1997

Structure and chromosomal localization of the beta2 subunit of the human brain sodium channel.

The beta2 subunit of rat brain voltage-gated sodium channels modulates their cell-surface expression and gating. It is a 33 kDa glycoprotein with a single transmembrane segment and an immunoglobulin-like fold resembling those of cell adhesion molecules in the extracellular domain. Here we report the cDNA sequence and genomic localization of the gene encoding the human beta2 subunit. The mature human beta2 protein has 89% amino acid sequence identity to rat beta2 and has four conserved consensus sites for N-linked glycosylation. The extracellular cysteine residues which are predicted to form the disulfide linkage in the immunoglobulin-like fold are also conserved, indicating that the overall structure is preserved between these two species. Using fluorescent in situ hybridization (FISH), we localized the beta2 gene to human chromosome 11q3. Mutations in this region of chromosome 11 in Charcot-Marie-Tooth syndrome type 4B and in other neurological diseases cause abnormal myelination and neurological deficits. The similarity of beta2 to cell adhesion molecules, including myelin protein p0, and its chromosomal location at 11q23 suggest a potential role in these demyelinating diseases.

Amino Acid Sequence↗
PubMed1997

Predicting RNA structures: the model of the RNA element binding Rev meets the NMR structure.

BACKGROUND: How accurate are the predictions of RNA three-dimensional structures? Assessing this accuracy requires the detailed comparison of the prediction with the experimentally determined structure. Previously, sequence variation in RNA aptamers that bind the Rev protein was used to infer a three-dimensional model of the Rev-binding element (RBE) RNA. Although much of this model has been substantiated by subsequent experimental data, its validity remains to be determined by confronting it with the structure determined by NMR spectroscopy. RESULTS: A series of different criteria such as geometric parameters (root mean square deviation, interproton distances, torsions and puckering), helicoidal parameters (base pairing and base stacking) and stability considerations (conformational energies) have been evaluated to identify common and distinguishing structural characteristics of the model and the NMR structure. CONCLUSIONS: The detailed comparison of the two structures reveals striking structural similarities at both the global and local level that validate the RNA modeling approach that we have used. Analysis of the structural differences and the precision of the model suggest that the limitations of the method are related to the amount of structural information available for modeling.

Binding Sites↗
PubMed1997

The effect of losigamone (AO-33) on electrical activity and excitatory amino acid release in mouse cortical slices.

1. Losigamone is a novel anticonvulsant the mechanism of action of which is not known. This study investigated the effect of losigamone on spontaneous, NMDA- and AMPA-induced depolarizations in the cortical wedge preparation of the DBA/2 mouse (which are susceptible to sound-induced seizures) and on endogenous amino acid release from BALB/c mouse cortical slices. 2. Cortical wedges exhibit spontaneous depolarizations in magnesium-free medium and losigamone was effective in significantly reducing these spontaneous depolarizations at concentrations of 100 microM and above. 3. NMDA-induced depolarizations were significantly reduced by losigamone at concentrations of 25 microM and above. Losigamone had no effect on AMPA-induced depolarizations. 4. Veratridine (20 microM) and potassium (60 mM) were used to stimulate the release of amino acids from mouse cortex. Veratridine-stimulated release of glutamate was significantly reduced by losigamone at concentrations of 100 microM and above, while potassium-stimulated release was significantly reduced by losigamone at 200 microM. 5. NMDA antagonism and inhibition of excitatory amino acid release may contribute to the anticonvulsant effect of losigamone.

Animals↗
PubMed1997

The HSD-hCG vaccine prevents pregnancy in women: feasibility study of a reversible safe contraceptive vaccine.

PROBLEM: To develop a vaccine for reversible control of fertility in women. MATERIALS AND PROTOCOLS: Purified beta subunit of hCG annealed to purified alpha subunit of ovine LH linked chemically to tetanus toxoid (TT) and diphtheria (DT); vaccine employed at 300 micrograms gonadotropin equivalent per injection adsorbed on alhydrogel with 1 mg SPLPS added in the first injection; Phase I safety trials in 47 women with elective tubal ligation; Phase II efficacy studies in 148 proven fertile women (2 children), sexually active, desirous of family planning using IUD; IUD removed when anti-hCG titres exceed 50 ng/ml hCG bioneutralization capacity; boosters given to maintain above threshold antibody levels; post coital tests conducted in 8 volunteers; sera of protected women analysed for immuno-determinants recognized by competitive enzyme immunoassays employing a panel of monoclonal antibodies and by direct binding to synthetic peptides; recombinant vaccines expressing beta hCG as a secreted product or as a fused protein anchored on membrane. RESULTS: Immunization was well tolerated with no significant changes in endocrine, metabolic and hematological indices. Normal ovulatory cycles were maintained as indicated by menstrual regulation. The vaccine was highly effective in preventing pregnancy (1 pregnancy in 1224 cycles ) at and above antibody titres of 50 ng/ml. Antibodies declined in course of time in absence of boosters, with conceptions occurring below 35 ng/ml titres indicating regain of fertility. Ability of antibodies to prevent pregnancy was confirmed by post coital tests. High avidity (10(10) M-1) and other characteristics of antibodies generated by the vaccine are described and compared with those induced by two other hCG vaccines having undergone Phase I trials. The antibody response of the HSD vaccine in humans is characterized predominantly to an epitope recognized by the monoclonals 206 and P3W80. The antibodies had low or no reactivity with the carboxy terminal peptide and 38-57 region peptide. Live recombinant vaccines expressing beta hCG as a membrane anchored peptide generated antibody response to hCG in all animals following a single injection. CONCLUSIONS: Reversible fertility control is feasible with the HSD-hCG vaccine without impairment of ovulation or disturbance of menstrual regularity. Suggestions have been made for further optimization of the vaccine, which include replacement of TT and DT by a panel of T non B determinants communicating with the entire MHC spectrum and development of recombinant vaccine expressing beta hCG along with membrane anchored carrier.

Animals↗
PubMed1996

Effects of felbamate on veratridine- and K(+)-stimulated release of glutamate from mouse cortex.

Felbamate is a novel anticonvulsant which may modulate the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor complex. This study examined the effect of felbamate and 5,7-dichlorokynurenic acid on veratridine (20 microM)- and K+ (60 mM)-stimulated release of amino acids in mouse cortical slices. Felbamate significantly decreased veratridine-induced release of glutamate at 400 microM and 800 microM but had no effect on K(+)-stimulated release. 5,7-Dichlorokynurenic acid had no effect on amino-acid release in concentrations up to 200 microM. The inhibitory effect of felbamate on veratridine-induced release of glutamate may be due to inactivation of voltage-sensitive Na+ channels.

Animals↗
PubMed1996

p53 expression in four human medulloblastoma-derived cell lines.

p53 is a tumor suppressor gene found on the short arm of chromosome 17. Loss of one p53 allele and alteration of the other is found in a variety of tumors, including highgrade glial tumors. Point mutations in the remaining allele occur in a highly conserved region of the gene encompassing exons 5-8. Although 40-50% of medulloblastomas lose sequences on the short arm of chromosome 17, alteration of p53 in these tumors is infrequent. To further characterize genetic alteration of p53 in medulloblastoma, we performed a mutational analysis of four medulloblastoma-derived cell lines established by our laboratory. Using two variable-number tandem repeat markers which map distally to p53, we found evidence indicating loss of sequences on the distal end of chromosome 17p in all four lines. However, no gross alterations of the p53 gene were detected. Northern analysis revealed expression of equivalent amounts of full-length p53 messenger RNA in each cell line. Using the polymerase chain reaction to amplify exons 5-8 of the p53 gene, we directly sequenced the amplified fragments and detected no mutations in any of the cell lines. Our results demonstrate that loss of p53 function through gene deletion and/or recessive mutation is not required for growth in our cell lines.

Alleles↗
PubMed1996

A docking and modelling strategy for peptide-RNA complexes: applications to BIV Tat-TAR and HIV Rev-RBE.

BACKGROUND: In spite of the great interest in the interaction between RNAs and proteins, no general protocol for modelling these complexes is presently available. This methodological vacuum is particularly acute because the structure of few such complexes is known. RESULTS: A general strategy for docking and modelling RNA-protein complexes has been developed. The docking procedure involves minimizing electrostatic and van der Waals' interaction energies of conformationally rigid structures during docking. After docking, libraries of amino acid sidechain conformations are searched to obtain the best interactions between the peptide and the RNA. Using this method, we have reproduced the structure of a bovine immunodeficiency virus (BIV) Tat peptide bound to BIV TAR RNA and have developed a model for the structure of the arginine-rich HIV-1 Rev peptide (Rev34-50) interacting with the Rev-binding element (RBE). CONCLUSIONS: The resulting model of the Rev34-50-RBE complex predicts that although no single arginine sidechain is responsible for complex formation, residues Arg2, Arg5 and Arg11 are more important for binding than the other arginine residues in the peptide. One model is supported by binding measurements performed on wild-type and mutant RBE molecules with the peptide.

Animals↗
PubMed1996

Transcranial Doppler in the evaluation of internal carotid artery dissection.

BACKGROUND AND PURPOSE: A subject with dissection of the internal carotid artery (ICA) may present with a variety of symptoms, from headache to stroke. Thus far, it has not been possible to identify the subset of patients at risk for cerebral ischemia. Because the majority of these ischemic events are secondary to embolic phenomena, we used transcranial Doppler (TCD) evaluation with emboli monitoring to study 17 consecutive patients with ICA dissection treated at Harborview Medical Center, Seattle, Wash, during a 2-year period from 1992 until 1994. METHODS: Ten patients with ICA dissection secondary to trauma and seven with spontaneous ICA dissection were diagnosed by carotid angiography and studied by TCD from the time of diagnosis through initiation of therapy. Emboli monitoring was performed in the middle cerebral artery (MCA) ipsilateral to the dissection at the initial evaluation and intermittently thereafter to ensure that the emboli stopped with treatment. RESULTS: Emboli were detected in the MCA distal to the dissection in 10 of 17 patients (59%). Patients with microemboli detected by TCD presented with a stroke (70%) much more frequently than those without emboli (14%) (P=.0498). The presence of a pseudoaneurysm did not increase the risk of either microemboli or stroke. CONCLUSIONS: We have demonstrated a high incidence of intracranial microemboli in the MCA distal to carotid dissections and a significant correlation between the presence of emboli and stroke. TCD can therefore be used as an adjunctive tool to manage patients with suspected carotid dissection and may prove useful in evaluating the efficacy of treatment in reducing microemboli and subsequent stroke.

Adolescent↗
PubMed1995

The design of peptide-based substrates for the cdc2 protein kinase.

The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].

Amino Acid Sequence↗
PubMed1995

Structure determination and analysis of local bending in an A-tract DNA duplex: comparison of results from crystallography, nuclear magnetic resonance, and molecular dynamics simulation on d(CGCAAAAATGCG).

We have presented a detailed analysis for structure determinations for the DNA duplex d(CGCAAAAATGCG) obtained from X-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulation. Each of the structures for the duplex deviates from the structure of the canonical form of B-DNA in a number of observable characteristics. Specifically, the three determinations all contain DNA axis deflections at the junctions of the A-tract with the flanking sequences. The analysis provided shows that the general characteristics of the structures obtained for d(CGCAAAAATGCG) from X-ray, NMR, and MD methods turn out to be quite similar. The extent to which this result can be generalized remains to be established by consideration of similar cross-comparisons on diverse oligonucleotide structures.

Base Sequence↗
PubMed1995

Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract.

Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine. Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions. A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium. Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions. Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella. Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine.

Acrosome↗
PubMed1995

The effect of the desglycinyl metabolite of remacemide hydrochloride (FPL 12495AA) and dizocilpine (MK-801) on endogenous amino acid release from mouse cortex.

1. In this study the effect of FPL 12495AA, the desglycinyl metabolite of remacemide hydrochloride and dizocilpine (MK-801), on potassium- and veratridine-stimulated release of neurotransmitter amino acids from mouse cortical slices was investigated. 2. Veratridine (20 microM) and potassium (60 mM) produced a preferential release of glutamate and aspartate. Potassium-stimulated release was calcium-dependent, while veratridine-stimulated release was only partially affected by removal of calcium from the medium. 3. FPL 12495AA significantly inhibited veratridine- and potassium-stimulated release of glutamate and aspartate. Lower concentrations of FPL 12495AA were needed to inhibit veratridine-stimulated release of glutamate (12.5 microM) than potassium-stimulated release (100 microM). 4. Dizocilpine significantly inhibited veratridine- and potassium-stimulated release of glutamate and aspartate at concentrations of 100 microM and above. 5. FPL 12495AA and dizocilpine both have an affinity for the ion channel subsite of the N-methyl-D-aspartate (NMDA) receptor. The reduction of potassium-stimulated release of glutamate and aspartate by FPL 12495AA and dizocilpine is probably due to NMDA receptor blockade. 6. FPL 12495AA inhibited veratridine-stimulated release at a concentration of 12.5 microM while dizocilpine was effective only at a concentration of 100 microM. This difference in efficacy is probably due to the higher affinity of FPL 12495AA compared to dizocilpine at the veratridine-binding site on the sodium channel.

Acetamides↗
PubMed1995

Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response.

Animals↗
PubMed1995

Duplex accuracy compared with angiography in the Veterans Affairs Cooperative Studies Trial for Symptomatic Carotid Stenosis.

Duplex ultrasonography is frequently used for noninvasive screening for extracranial occlusive disease. In a multicenter, prospective, randomized trial for carotid endarterectomy in symptomatic patients, the duplex ultrasound examination was compared with conventional angiographic findings for internal carotid artery (ICA) stenosis in 178 patients. Angiograms were interpreted by radiologists both at local medical centers and at a central site; the angiographic determination of ICA stenosis was calculated as percent diameter at the point of maximal narrowing compared with the normal distal ICA. Comparisons were made for 328 arteries, including both the symptomatic (> 50% stenosis determined by angiography) and the asymptomatic (variable degrees of stenosis) sides. Duplex ultrasonography sensitivity varied from 0.24 for 30 to 49% stenosis to 0.71 for 50 to 79% stenosis and 0.91 for ICA occlusion. Using a 50% stenosis cutpoint, duplex ultrasonography sensitivity was 0.90 with a specificity of 0.76. Duplex scan readings underestimated the degree of stenosis in the 30 to 49% stenosis group in 48% of the cases. There was no apparent relationship between the accuracy of stenosis determinations and that of external carotid artery stenosis, carotid plaque morphology, or ulceration determinations by ultrasound. On the basis of the benefit provided by carotid endarterectomy in symptomatic patients with high-grade lesions, duplex ultrasound accuracy is essential if noninvasive testing is used to make clinical decisions. In situations in which duplex findings may not be reliable, such as in the mild-to-moderate stenosis and occlusion categories, carotid angiography may be indicated.

Angiography↗
PubMed1995

A guide to placement of parietooccipital ventricular catheters. Technical note.

Accurate placement of parietooccipital ventricular catheters can be difficult and frustrating. To minimize the morbidity of the procedure and lengthen the duration of shunt function, the catheter tip should lie in the ipsilateral frontal horn. The authors describe a posterior ventricular guide (PVG) for placement of parietooccipital catheters that operates by mechanically coupling the posterior burr hole to the anterior target point. In a series of 38 patients who underwent ventriculoperitoneal shunting with the assistance of the guide, postoperative computerized tomography (CT) scanning revealed that 35 (92.0%) had accurate catheter placement. In comparison, a retrospective review of free-hand posterior catheter placement revealed good catheter position in only 22 of 43 patients (51.1%). The use of the guide added less than 5 minutes to the entire procedure, and there were no complications related to its use. The PVG is a simple and useful tool that aids in the placement of parietooccipital ventricular catheters.

Adolescent↗
PubMed1995

Establishment and characterization of four human medulloblastoma-derived cell lines.

We have established four cell lines, UW228-1, UW228-2, UW228-3 and UW443, from two posterior fossa medulloblastomas. The three UW228 sublines originated from a tumor with a diploid DNA content, while the tumor of origin of UW443 was predominantly tetraploid. Both tumors displayed areas of immunopositivity for synaptophysin and glial fibrillary acidic protein. All four cell lines have been grown as monolayers in continuous culture for 50 to 200 passages, are not contact inhibited at high density, and form colonies in soft agar. The UW228 sublines are aneuploid, have similar modal chromosome numbers, similar chromosomal duplications and identical marker chromosomes, and display loss of heterozygosity for identical sequences at the distal end of chromosome 17p. UW443 is diploid and also shows loss of heterozygosity for a distal sequence on chromosome 17p. All lines are immunopositive for two or more neurofilament proteins, three lines (UW228-1, UW228-2 and UW443) are immunopositive for synaptophysin, and none are immunopositive for glial fibrillary acidic protein. The lines differ in sensitivity to the alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. They also differ in dependence on the DNA repair protein O6-methylguanine-DNA methyltransferase for alkylating agent resistance and in levels of the DNA repair activities apurinic/apyrimidinic endonuclease and DNA polymerase beta. These properties establish UW228-1, UW228-2, UW228-3 and UW443 as four new, phenotypically distinct medulloblastoma-derived cell lines.

Antineoplastic Agents, Alkylating↗