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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 1,189 records · Page 66Linked to original sources
PubMed1994

Muscarinic acetylcholine receptor subtypes in rat visual cortex--a comparative study using quantitative receptor autoradiography and in situ hybridization.

The laminar pattern of M1- and M2-muscarinic acetylcholine receptors (mAChR) in rat visual cortex has been compared with the distribution of the corresponding m1, m2, m3 and m4 receptor genes using both quantitative receptor autoradiography and in situ hybridization histochemistry. The laminar distribution of 3H-pirenzepine binding to M1-mAChRs in rat visual cortex shows a bimodal pattern with higher binding levels in upper layer III and deeper layer VI. In contrast, highest binding of 3H-AF-DX384 to M2-mAChRs was observed in upper layer IV (100%) and upper layer VI (about 80% of highest binding). The m1 receptor mRNA is almost homogeneously distributed throughout the visual cortex, whereas the m2mAChr mRNA predominates in layer IV with lower levels in layers I and V. The highest amounts of m3mAChR mRNA in rat visual cortex were observed in layer II, while the distribution of m4mAChR transcripts shows a bimodal pattern with peaks in layers III and upper layer VI. The distinct laminar pattern of mRNA muscarinic receptor subtypes in rat visual cortex suggest specific roles of the muscarinic receptor in visual function.

Animals↗
PubMed1994

Population structure of goat biting louse Bovicola caprae (Phthiraptera: Ischnocera).

The population structure of Bovicola caprae on goats has been recorded by examining 57 goatskins (45 by brushing cum searching and 12 by dissolving method). Population composition, sex ratio, adult nymph ratio has been recorded at different levels of infestation. The female dominated over the male population and male: female has been found to be 1:2 by brushing cum searching and 1:1.7 by dissolving method. Likewise, A:N was found to be 1:1.2 by brushing cum searching and 1:17 by dissolving method. The number of males and females follow similar pattern throughout population and were found to exhibit a high degree of correlation.

Animals↗
PubMed1994

Expression and assembly of procoagulant complexes by human pleural mesothelial cells.

Many pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor VIIa to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

Blood Coagulation Factors↗
PubMed1994

Pediatric clerkship performance in diverse community clinical settings.

OBJECTIVE: To determine whether multiple and diverse clinical settings and resources in a pediatric clerkship result in satisfactory performance by medical students and affect their choice of a pediatric residency after graduation. DESIGN: Retrospective study 1985 through 1990. SETTING: Medical students in a pediatric clerkship in a medical school that is part of a land grant university where the clinical curriculum is conducted in six geographically distinct communities. MAIN OUTCOME MEASURE: (1) Three performance criteria including: multiple choice final examination score, clinical performance evaluations by preceptors, and formal evaluation of patient written records. (2) Students matching with a pediatric or combined internal medicine/pediatric residency. RESULTS: Of 499 students 92.8% successfully passed all three criteria with no statistically significant differences between communities. A smaller percentage of students (15.2%) (community range, 6 to 18.5%) entered straight or combined pediatric residencies, whether or not a pediatric residency was present in the community. CONCLUSIONS: Medical students can achieve satisfactory competency in pediatric clerkships offered in diverse clinical settings, and choose pediatric residency training whether or not they are exposed to a pediatric residency during their clerkship. A problem-based learning curriculum does not hinder selecting pediatrics as a career choice.

Career Choice↗
PubMed1994

Aspergillus ribotoxins react with IgE and IgG antibodies of patients with allergic bronchopulmonary aspergillosis.

Asp f I has been identified as a major allergen of Aspergillus fumigatus and shows amino acid sequence homology with the mitogillin family of cytotoxins. In this study we compared the various cytotoxins from Aspergillus species for their reactivity with immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies in the serum samples of patients with allergic bronchopulmonary aspergillosis. Elevated levels of IgE and IgG antibodies to all A. fumigatus antigens and ribotoxins were demonstrated in a majority of ABPA patients as compared with allergic patients with asthma and normal subjects. In approximately 75% of patients with allergic aspergillosis, IgE antibody to ribotoxins showed marked elevation, whereas in the remaining patients it was below the baseline, although crude A. fumigatus antigen showed higher specific serum IgE. These results suggest that cytotoxins play a major role in the pathogenesis of allergic bronchopulmonary aspergillosis.

Antibodies↗
PubMed1993

Structure/function studies of HIV-1(1) reverse transcriptase: dimerization-defective mutant L289K.

Virion-derived HIV-1 reverse transcriptase (RT) has subunits of molecular mass 66 and 51 kDa (p66 and p51, respectively) in an approximately 1:1 ratio. Since enzyme activity appears to depend on dimerization of these subunits, identification of critical regions of primary sequence required for proper dimerization could lead to potential targets for antiviral therapy. A central region of primary sequence contains a leucine hepta-repeat motif from leucine 282 to leucine 310 that has been suggested to be involved in dimerization [Baillon, J. G., Nashed, N. T., Kumar, A., Wilson, S. H., & Jerina, D. M. (1991) New Biol. 3, 1015-1019]. A region including this hepta-repeat was recently shown to be involved in protein-protein interactions required for dimerization [Becerra, S. P., Kumar, A., Lewis, M. S., Widen, S. G., Abbotts, J., Karawya, E. M., Hughes, S. H., Shiloach, J., & Wilson, S. H. (1991) Biochemistry 30, 11708-11719]. To investigate the role of this repeat motif in dimerization, we performed site-directed mutagenesis of these leucine residues from position 282 to position 310. Mutations were introduced into p66 and p51 RT coding sequences, and the individually purified RT subunit polypeptides were compared with wild-type polypeptides for dimerization. Physical characterization of the purified mutant peptides was conducted by circular dichroism analysis. Binding between p66 and p51 was studied by gel filtration, ultracentrifugation, and CD analysis. L289K-p66 was unable to dimerize with itself and wild-type or L289K-p51. The leucine repeat motif in the p66 subunit appears to be critical in formation of the heterodimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Molecular dynamics with weighted time-averaged restraints for a DNA octamer. Dynamic interpretation of nuclear magnetic resonance data.

For conformationally flexible molecules in solution, NMR-derived distance restraints are time-averaged. In contrast to deriving structures from NMR distance constraints via conventional restrained molecular dynamics (rMD), the range of conformational flexibility may be better represented by MD simulations using weighted time-averaged restrains (MD-tar). This approach has been utilized for dynamic structure refinement of the DNA octamer [d(GTATAATG].[d(CATATTAC)] which contains the Pribnow box consensus sequence. An improved set of distance restraints was calculated via complete relaxation matrix analysis utilizing the solution structure of the octamer which was determined previously by rMD as a starting model. MD-tar calculations were performed with the program AMBER4 under various conditions, some including explicit solvent. All trajectories generated via MD-tar exhibited reduced constraint energies and average deviations for the distances compared to standard rMD. Quantitative comparison with experimental data, i.e. two-dimensional NOE intensities and COSY-derived coupling constants yielded a significant improvement for MD-tar simulated structures relative to rMD-derived structures. The conformational envelope of the MD-tar structures is wider than that from rMD and even unrestrained MD. Explicit solvent force-fields tightened the conformational envelope, leading to even better agreement with experimental data. All MD-tar simulations exhibit sugar repuckering for basically all residues, yielding a minor population in the low N-region and one or more S-conformers. For most backbone torsion angles, one or more minor conformers were found, while the major conformations generally coincided with those obtained in standard rMD. Distributions of helical parameters for MD-tar trajectories are rather symmetric but slightly broader than those for rMD. Average values and associated standard deviations are discussed with respect to sequence-dependent variations. All trajectories obtained with an explicit solvent force field exhibited a narrower minor groove compared to in vacuo calculations.

Carbohydrate Conformation↗
PubMed1993

HIV-1 reverse transcriptase: inhibition by 2',5'-oligoadenylates.

2',5'-Oligoadenylates (2-5A) and derivatives are noncompetitive inhibitors of primer/HIV-1 reverse transcriptase complex formation. The mechanism and specificity of this inhibitory action of 2-5A and 2-5A derivatives have been evaluated with 2-5A molecules modified in ribosyl moiety, chain length, extent of 5'-phosphorylation, and 2',5'-phosphodiester linkage. UV covalent cross-linking of preformed complexes of p66/p66 homodimer or p66/p51 heterodimer recombinant HIV-1 reverse transcriptase and the primer analog pd(T)16 allowed analysis of the initial step in HIV-1 reverse transcriptase-catalyzed DNA synthesis. Utilizing this primer binding assay, it is demonstrated that 2-5A and 2-5A derivatives inhibit the binding of pd(T)16 to HIV-1 reverse transcriptase. This inhibition is specific for the 2',5'-internucleotide linkage in that the corresponding 3',5'-adenylate derivatives do not exhibit inhibitory activity. Enhanced inhibitory properties were observed following modifications of the 2-5A molecule which result in an increase in hydrophobicity. Replacement of the D-ribosyl moiety of 2-5A with the 3'-deoxyribosyl moiety increased the inhibition of primer/HIV-1 reverse transcriptase complex formation 15-20%. 2',5'-Phosphorothioate substitution yielded the most effective inhibitors, with Ki's of 7-13 microM. In all cases, inhibition of primer/HIV-1 reverse transcriptase complex formation showed a preference for the 5'-triphosphate moiety. Nonphosphorylated derivatives were not inhibitory; 5'-monophosphate derivatives exhibited little or no inhibition. The inhibition of primer binding to HIV-1 reverse transcriptase correlated well with the inhibition of DNA-directed DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1993

Threonine synthase of Escherichia coli: inhibition by classical and slow-binding analogues of homoserine phosphate.

L-threo-3-Hydroxyhomoserine phosphate, derived from the antimetabolites L-threo-3-hydroxyaspartate and L-threo-3-hydroxyhomoserine [Shames, S. L., Ash, D. E., Wedler, F. C., and Villafranca, J. J. (1984) J. Biol. Chem. 258, 15331-15339], is a classical competitive inhibitor of threonine synthase (Ki = 6 microM) with structural elements of both substrate and product. L-2-Amino-5-phosphonovaleric acid also inhibits the enzyme competitively with a Ki (31 microM), comparable to Km for L-homoserine phosphate. In contrast, a structural analogue of Hse-P, L-2-amino-3-[(phosphonomethyl)thio]propionic acid exhibits a Ki = 0.11 microM (ca. 100-fold less than Km for L-Hse-P), along with "slow, tight" inhibition kinetics. Nuclear magnetic resonance was used with these inhibitors to probe for pyridoxal phosphate-catalyzed hydrogen-deuterium exchange reactions characteristic of substrates. With L-threo-3-hydroxy-homoserine phosphate, H-D exchange occurs only at the C-alpha position, but for homoserine in the presence of phosphate and for L-2-amino-5-phosphonovaleric acid and L-amino-3[(phosphonomethyl)thio]propionic acid (APMTP), H-D exchange occurs at C-alpha and stereospecifically at C-beta. For L-homoserine plus phosphate and L-2-amino-5-phosphonovaleric acid, the rate of H-D exchange at C-alpha is 8-45 times faster than at C-beta. For L-2-amino-3-[(phosphonomethyl)thio]propionic acid, the C-alpha to C-beta exchange rate ratio is near unity, due to a 700-fold decrease in the C-alpha rate for the analogue. Taken with information from molecular modeling, these data can be interpreted in terms of the current working hypothesis for the catalytic mechanism. Specifically, the slow, tight inhibition by APMTP results from its being carried further into the catalytic cycle than other analogues prior to forming an intermediate that is blocked from further catalysis.

2-Amino-5-phosphonovalerate↗
PubMed1993

Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.

Amino Acid Sequence↗
PubMed1993

Cloning of cDNA for the alpha subunit of mouse insulin-like growth factor I receptor and the role of the receptor in metanephric development.

Various growth factors influence mammalian development by binding to specific cell surface receptors. These interactions are followed by a series of intracellular transductional events leading to a wide variety of biological effects. To establish the role of insulin-like growth factor I receptor (IGF-IR) in renal development, cDNA for the alpha subunit of the mouse IGF-IR was isolated, characterized, and used in expression studies and antisense experiments in a metanephric organ culture system. A 989-bp insert, encoding the signal peptide and 299 amino acids, isolated from a newborn mouse kidney cDNA library had 99% and 91% homology with the nucleotide sequences encoding the rat and the human IGF-IR, respectively. An approximately 11-kb message was readily detected by Northern blot analysis of RNA from the developing kidney at day 13 of gestation, and it declined during the subsequent embryonal and neonatal periods. In situ hybridization revealed high levels of message over the ureteric bud and its branches. A lower level of message was seen in the neonatal kidney, confined mainly to the tubules. Antisense oligodeoxynucleotide-treated metanephric kidneys were reduced in size and had a decreased population of nephrons with marked disorganization of ureteric bud branches. Immunofluorescence studies indicated an arrest of IGF-IR translation after antisense exposure. Immunoprecipitation studies showed a marked decrease in the biosynthesis of various extracellular matrix proteins that serve as regulators of morphogenesis. These studies suggest that the nucleotide sequence encoding the alpha subunit of mouse IGF-IR is highly conserved and that the receptor might play an essential role in the organogenesis of the kidney.

Aging↗
PubMed1993

Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking.

Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains. The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not. To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity. Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P. Karpel, R. L., and Wilson, S. H. (1990b) J. Biol. Chem. 265, 2124-2131; Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) J. Biol. Chem. 266, 19618-19625). Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to [32P] p(dT)16. Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58. Sequence analysis of the various [32P]p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe.

Amino Acid Sequence↗
PubMed1993

Specific molecular interaction sites on factor VII involved in factor X activation.

Factor VII, a serine-protease zymogen, and tissue factor, the cellular receptor/coenzyme, are the protein components of the macromolecular complex which initiates the extrinsic pathway of the coagulation cascade. Previous studies were directed to the identification of functional sites on factor VII which mediate factor X activation, employing a series of potentially inhibitory synthetic peptides representing the primary structure of factor VII and antibodies to selected peptides. The involvement of at least four high-affinity interactive regions [factor VII (44-50), (196-229), (285-305) and (376-396) peptides] on the surface of factor VII was clearly demonstrated. The minimal sequences for the expression of inhibitory activity of these four molecular recognition domains on factor VII were identified using short and overlapping peptides. The short factor VII-(206-218)-peptide (most inhibitory peptide in the sequence 196-229 on factor VII) inhibited the binding of factor VII to the tissue-factor-expressing J82 cell line. Furthermore, radiolabeled [Tyr201] factor VII-(199-221)-peptide, with a tyrosine substituted for the normal tryptophan residue, was specifically bound to J82 cells, and also the binding of the radiolabeled peptide to this cell line was specifically inhibited by a monoclonal antibody to tissue factor, confirming that the interaction site for tissue factor on factor VII is present within the peptide sequence 196-229. Kinetic analyses suggested that the regions represented by factor VII-(285-305)- and factor VII-(376-396)-peptides are involved in factor X recognition and the chemical cross-linking of the radiolabeled peptides resulted in specific binding to factor X, confirming that these two regions on factor VII represent the substrate-recognition site. Furthermore, these radiolabeled peptides specifically interact with the heavy chain of factor X, suggesting that the complementary binding region for the substrate-recognition site on factor VII are present on the heavy chain of factor X.

Amino Acid Sequence↗
PubMed1993

Alpha 4 beta 1-integrin expression on sickle reticulocytes: vascular cell adhesion molecule-1-dependent binding to endothelium.

Important complications in sickle cell anemia occur secondary to vascular occlusion, which is postulated to be initiated by interactions of erythrocytes with vascular endothelial cells. In patients with sickle cell anemia, up to 25% of reticulocytes express the alpha 4 beta 1-integrin complex. Furthermore, erythrocytes from patients with sickle cell anemia bind to endothelial cells activated by tumor necrosis factor alpha via (TNF alpha) via interactions between erythrocyte alpha 4 beta 1 and endothelial cell vascular cell adhesion molecule-1 (VCAM-1). Thus, binding of alpha 4 beta 1-expressing reticulocytes to cytokine-activated endothelial cells may initiate vascular complications in sickle cell anemia and perhaps other hemolytic anemias during episodes of infection and inflammation.

Anemia, Sickle Cell↗