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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methylpyrazole.

Alcohol Dehydrogenase↗
PubMed1992

Mammalian DNA polymerase beta: characterization of a 16-kDa transdomain fragment containing the nucleic acid-binding activities of the native enzyme.

The 39-kDa DNA polymerase beta (beta-Pol) molecule can be readily converted into two constituent domains by mild proteolysis; these domains are represented in an 8-kDa N-terminal fragment and a 31-kDa C-terminal fragment [Kumar et al. (1990a) J. Biol. Chem. 265, 2124-2131]. Intact beta-Pol is a sequence-nonspecific nucleic acid-interactive protein that binds both double-stranded (ds) and single-stranded (ss) polynucleotides. These two activities appear to be contributed by separate portions of the enzyme, since the 31-kDa domain binds ds DNA but not ss DNA, and conversely, the 8-kDa domain binds ss DNA but not ds DNA [Casas-Finet et al. (1991) J. Biol. Chem. 266, 19618-19625]. Truncation of the 31-kDa domain at the N-terminus with chymotrypsin, to produce a 27-kDa fragment (residues 140-334), eliminated all DNA-binding activity. This suggested that the ds DNA-binding capacity of the 31-kDa domain may be carried in the N-terminal segment of the 31-kDa domain. We used CNBr to prepare a 16-kDa fragment (residues 18-154) that spans the ss DNA-binding region of the 8-kDa domain along with the N-terminal portion of the 31-kDa domain. The purified 16-kDa fragment was found to have both ss and ds polynucleotide-binding capacity. Thermodynamic binding properties for these activities are similar to those of the intact enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

DNA polymerase beta and DNA synthesis in Xenopus oocytes and in a nuclear extract.

The identities of the DNA polymerases required for conversion of single-strand (ss) M13 DNA to double-strand (ds) M13 DNA were examined both in injected Xenopus laevis oocytes and in an oocyte nuclear extract. Inhibitors and antibodies specific to DNA polymerases alpha and beta were used. In nuclear extracts, inhibition by the antibody to polymerase beta could be reversed by purified polymerase beta. The polymerase beta inhibitors, dideoxythymidine triphosphate (ddTTP) and dideoxycytidine triphosphate (ddCTP), also blocked DNA synthesis and indicated that polymerase beta is involved in the conversion of ssDNA to dsDNA. These results also may have particular significance for emerging evidence of an ssDNA replication mode in eukaryotic cells.

Animals↗
PubMed1992

A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3.

Following an ill-defined activation event, the Arg-Gly-Asp (RGD) recognition site of the platelet integrin, glycoprotein IIb-IIIa (alpha IIb beta 3), can bind to fluid-phase, RGD-containing protein ligands, such as fibrinogen, or to the murine monoclonal IgM, PAC-1, which contains the sequence Arg-Tyr-Asp (RYD) within the third complementarity-determining region of its heavy chain (H3). PAC-1 has thus become a widely exploited marker of platelet alpha IIb beta 3 activation. In this report, we compare PAC-1 with two murine IgG, OP-G2 (IgG1 kappa) and LJ-CP3 (IgG1 kappa), that also contain the sequence RYD in H3 but bind to alpha IIb beta 3 without prior activation. Each antibody can inhibit the binding of the other two to intact platelets or to purified IIb-IIIa, the binding of each antibody is completely inhibited by peptides containing RGD, and H3 of each antibody uses the germline D-gene DSP 2.10 (CTATAGGTACGAC) which includes the sequence RYD. Two other murine IgG, HP20 and PCG1-1, cloned and sequenced by other laboratories, also utilize the DSP 2.10 sequence, but neither antibody binds to alpha IIb beta 3. From a comparison of the H3 sequences of these antibodies, we have developed a molecular model of the H3 loop region which can explain these differences in specificity. This model predicts that both the ability to bind to alpha IIb beta 3 and the activation dependence of that binding are a function of the orientation and, therefore, accessibility of the RYD sequence. This model and refinements thereof can be exploited to study the molecular basis for specificity and affinity of RGD-containing ligands for integrins.

Amino Acid Sequence↗
PubMed1992

Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants.

We have used the polymerase chain reaction to isolate fragments of Ty1-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Ty1-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Ty1-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Ty1-copia group retrotransposons within plant lineages has been a major factor in the evolution of Ty1-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.

Amino Acid Sequence↗
PubMed1992

Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase.

The double-stranded RNA (dsRNA)-binding domain of the human p68 kinase has been localized to the N-terminal half of the enzyme by using progressive deletion analysis and in vitro binding assays. To further define the domains responsible for binding to dsRNA, we cloned the mouse dsRNA-activated p65 kinase and used sequence alignment to identify conserved domains in the N-terminal region. Deletions in either of two 12-amino-acid-long and arginine- or lysine-rich regions abrogated binding to dsRNA. Moreover, in an in vivo growth inhibition assay in the yeast Saccharomyces cerevisiae, these mutants failed to exhibit a slow-growth phenotype.

Amino Acid Sequence↗
PubMed1992

Design and synthesis of a self-assembling peptide derived from the envelope proteins of HIV type 1. An approach to heterovalent immunogens.

A chimeric peptide that included sequences from gp120 and gp41 of HIV type 1 was synthesized. Cleavage from solid support yielded a composite of self-oligomerized products with molecular masses ranging from 5 to about 9 kDa. The oligomer but not its reduced, monomeric form was recognized by human anti-HIV sera and at least one of the two lysines in the sequence was involved in antibody binding. The oligomeric peptide was immunogenic, yielding a conformation-specific antibody response. Co-oligomerization of a hepatitis B surface Ag-derived peptide and the HIV type 1-derived peptide yielded a bivalent product in which conformational integrity of the individual components was maintained. Immunization with this hybrid peptide resulted in conformation-specific antibodies to both epitopes in all four murine strains tested. Lymphocyte proliferation assays revealed that the T epitopes resident in both peptide sequences remained active in the hybrid peptide. These results demonstrate the potential of this approach in generating multi- and heterovalent immunogens which may eventually find application as vaccines.

Amino Acid Sequence↗
PubMed1992

"Universal" T helper cell determinants enhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide.

Synthetic peptide constructs containing a limited number of epitopes are being currently investigated as subunit vaccines against a variety of pathogens. However, because of widespread nonresponsiveness to most such constructs, possibly attributable to MHC restriction, the choice of appropriate carrier molecules to enhance immunogenicity of peptides constitutes an important and essential aspect of designing synthetic immunogens for human use. Widely used vaccines such as tetanus toxoid (TT) have not been uniformly effective as carrier proteins because of the phenomenon of epitope-specific suppression in which induction of an immune response against a synthetic peptide conjugated to TT is prevented by preexisting immunity to TT. Recently, T cell determinants that can be recognized in the context of several class II MHC molecules have been identified in tetanus toxin as well as in the circumsporozoite protein of a human malarial parasite, Plasmodium falciparum. Such determinants can be potentially used to circumvent the problem of epitope-specific suppression. In the present study we evaluated two such T cell determinants, viz., tt830-844 from tetanus toxin and CST3 from the malarial parasite, for their ability to help induce a boostable antibody response and to overcome genetic nonresponsiveness to a synthetic 20-residue construct containing a B cell and an overlapping T cell epitope from a major merozoite surface protein of P. falciparum. Our data provide support for the view that widely recognized T cell determinants may be used as universal carrier molecules for general vaccination.

Adolescent↗
PubMed1992

Low-molecular weight B cell growth factor (BCGF-12KD) as an autocrine growth factor in B cell chronic lymphocytic leukemia.

The role of cytokines in the growth and spreading of human hematologic malignancies has been underlined in recent years. Here we report evidence that a human 12-kDa B cell growth factor (BCGF-12KD) may function as a growth stimulatory factor in B cell chronic lymphocytic leukemia (B-CLL). First, recombinant BCGF-12KD induced dose-dependent DNA synthesis in neoplastic B cells of four B-CLL patients tested. Second, seven different B-CLL clones secreted a BCGF-12KD-like product that accounted for their proliferation. A neutralizing monoclonal antibody (mAb; Ac8) directed against a BCGF-12KD synthetic peptide inhibited the spontaneous growth of the leukemic B cells. The same mAb blocked DNA synthesis in normal tonsillar B lymphocytes induced by the culture supernatant of spontaneously proliferating B-CLL cells. Finally, BCGF-12KD mRNA was expressed in freshly isolated (two of three patients) as well as in vitro proliferating B-CLL cells (three of three patients). These findings strongly suggest that BCGF-12KD can modulate the growth of B-CLL cells in vivo as well as in vitro. They may offer significant insights into the biology of this frequent B lymphoproliferative disorder.

Adult↗
PubMed1992

Hypersymmetry in a transcriptional terminator of Escherichia coli confers increased efficiency as well as bidirectionality.

Rho-independent transcriptional terminators in Escherichia coli usually consist of a GC-rich region encoding a sequence which allows the nascent transcript to form a stem-loop structure, and thereby apparently causes the RNA polymerase to pause; followed by an A-rich region on the DNA template strand, whose weak pairing to the consequently U-rich 3'-tail of the transcript is believed to aid release of the RNA. Quite commonly there is additional symmetry encoding, in the RNA, an A-rich sequence complementary to the 'tail', just upstream of the GC-rich motif. It has been pointed out by others that this should allow the termination of transcription in both directions. We hypothesized that it might also increase the efficiency of termination, for example by competing with the template DNA strand so as to help 'unzip' the 3'-end of the RNA from its complementary DNA. We have tested this hypothesis by deletion analysis of the hypersymmetric alpha-operon terminator, tL17. The results show that efficiency as well as bidirectionality is indeed adversely affected by deletion of the upstream A-rich sequence.

Base Sequence↗
PubMed1992

Infection with hepatitis A, B, delta, and human immunodeficiency viruses in children receiving cycled cancer chemotherapy.

Serological markers of hepatitis A, B, and Delta and human immunodeficiency viruses were studied in 25 children receiving cancer chemotherapy. Eighty-eight percent had pre-existing HAV immunity which was unaltered by chemotherapy. HDV infection was observed in 8% while HIV was conspicuous by its absence. Active HBV infection, observed in 76% of the children, was asymptomatic in the majority and was accompanied by a high incidence of HBe antigenaemia (57.9%) and its persistence. Pre-existing anti-HBs failed to prevent HBV infection recurrence, which was, however, transient and self-limiting. Multiple blood transfusions and repeated parenteral exposures appeared to be the possible sources of HBV acquisition. Transmission to close contacts was also observed. The study suggests that although HBV vaccine might not be protective against HBV infection in patients receiving cancer chemotherapy, it may prevent its persistence and thereby help in reducing chronic liver disease-related morbidity and a highly infectious reservoir. Strict HBV screening of blood donors, exclusive use of disposable equipment, and vaccination of close contacts of cancer patients is recommended, particularly in HBV endemic third-world countries.

Antineoplastic Agents↗
PubMed1992

Primary oesophageal carcinoma in teens.

Seven teenaged patients with primary oesophageal carcinoma, treated at our hospital in the last 6 years are presented and their management discussed. Five patients had localised disease. Three of them underwent total oesophagectomy, one was given radical radiotherapy, and one was lost to follow-up. Two patients had metastatic disease at presentation. The primary oesophageal carcinoma in this age group may not be so rare as reported in the literature and must be considered in the differential diagnosis of dysphagia. Like their older counterparts, the outcome depends on the stage of the disease and is not influenced by age per se.

Adenocarcinoma↗
PubMed1992

Expression and sequence analysis of cDNAs induced during the early stages of tuberisation in different organs of the potato plant (Solanum tuberosum L.).

cDNA clones of two genes (TUB8 and TUB13) which show a 25-30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.

Adenosylmethionine Decarboxylase↗
PubMed1992

Extreme heterogeneity of Ty1-copia group retrotransposons in plants.

We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants. All eight species studied contain reverse transcriptase fragments from Ty1-copia group retrotransposons. Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75%. Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila. The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants. Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species. We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.

Amino Acid Sequence↗
PubMed1992

Survival in patients with systemic lupus erythematosus in India.

Survival in patients with systemic lupus erythematosus (SLE) in developed countries has improved considerably over the last 2 decades. In India, however, survival in patients with SLE reported 5 years ago from this tertiary referral centre was very poor. The present study was conducted to examine trends, if any, towards improvement in the survival of patients with SLE attending the same centre during the period 1981-1990. No statistically significant change in survival was noted. It appears that improvement in the survival of SLE patients would require an overall improvement in the standard of medical care in India.

Adult↗
PubMed1992

Spinal anaesthesia with lidocaine 2% for caesarean section.

Spinal anaesthesia with 2, 2.5 or 3 ml of glucose-free lidocaine 2% was studied in 50 patients undergoing Caesarean section. Onset time, cephalad spread of analgesia, quality of analgesia, muscle relaxation, the cardiovascular effects and duration of analgesia and motor block were assessed. Reliable anaesthesia was provided with 2.5 and 3 ml while 2 ml of 2% lidocaine was insufficient. Onset time varied between 5.5 to 6 min and maximum cephalad spread was achieved in 10-15 min. The mean maximum extent of sensory analgesia was higher after 2.5 ml (T4.1) and 3 ml (T3.6) than after 2 ml (T7) (P < 0.001). Complete motor block was achieved in all the patients. The mean duration of sensory block was 123 +/- 6.23 min (2 ml) to 126 +/- 7.53 min (2.5 and 3 ml). The mean duration of motor block in 2.5 and 3 ml groups was higher (P < 0.001) than in the 2 ml group and was correlated with the dose of lidocaine (P < 0.05). Hypotension (SBP < 100 mmHg) was noted in 10% (n = 5) of patients in whom the cephalad spread of analgesia was also higher. All the neonates had an apgar score of 7 or more at 1 min. These results suggest that 2.5 to 3 ml of 2% lidocaine provides satisfactory anaesthesia for Caesarean section.

Adult↗
PubMed1992

Arterial blood nicotine concentration and coronary vasoconstrictive effect of low-nicotine cigarette smoking.

Low-nicotine cigarettes have been advertised to the public as less harmful to the cardiovascular system. We studied the effects of smoking two low-nicotine cigarettes on arterial and venous blood nicotine levels, hemodynamics, and coronary vascular tone in 12 patients referred for diagnostic coronary arteriography. All were chronic smokers as evidenced by their elevated baseline arterial and venous cotinine blood levels (139 +/- 30 ng/ml and 155 +/- 34 ng/ml, respectively). High-resolution coronary angiograms were evaluated "blindly" before and after smoking. An electronic caliper was used to measure the diameter of disease-free coronary segments of the left anterior descending and circumflex arteries. Arterial nicotine levels rose from 5 +/- 1 ng/ml at baseline to 37 +/- 7 ng/ml (p less than 0.01) after the first cigarette was smoked and to 45 +/- 8 ng/ml (p less than 0.01) after the second cigarette. Venous nicotine levels rose from 8 +/- 2 ng at baseline to 15 +/- 3 ng/ml (p less than 0.05) after the first cigarette and to 20 +/- 3 ng/ml (p less than 0.01) after the second cigarette. After the first cigarette heart rate increased 8 +/- 2 beats/min (p less than 0.003) and double product 1229 +/- 400 beats/min x mm Hg (p less than 0.02). Compared to baseline values, after the second cigarette heart rate increased 9 +/- 1 beats/min (p less than 0.001) and double product 1767 +/- 486 beats/min x mm Hg (p less than 0.01). Systolic, diastolic, and mean blood pressure did not change significantly after either the first or second cigarette.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac Catheterization↗