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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 1,099 records · Page 61Linked to original sources
PubMed1994

Identification of a novel determinant for basic domain-leucine zipper DNA binding activity in the acute-phase inducible nuclear factor-interleukin-6 transcription factor.

Nuclear factor-interleukin-6 (NF-IL6), a member of the CCAAT box/enhancer-binding protein (C/EBP) family, contains a basic domain-leucine zipper (bZIP) DNA binding motif. Controlled protease digestion was used to probe free and DNA-complexed NF-IL6 protein. Digestion with trypsin in the absence of DNA produced the leucine zipper domain (containing residues 303-345). In contrast, digestion of NF-IL6.DNA complexes produced a stable domain, spanning residues 266-345, termed the tryptic core domain (TCD). The NH2-terminal boundary of the TCD is longer than tryptic peptides reported from C/EBP alpha.DNA complexes. Digestion of NF-IL6 with endoprotease Asp-N produced a domain smaller than the TCD (NF-IL6 bZIP domains (NFBD) (272-345)), a domain identified either in the absence or the presence of DNA. Both recombinant peptides bind acute-phase response element DNA in a sequence-specific fashion. The equilibrium disassociation constant (Kd) for the TCD was 36 +/- 8 nM, whereas the Kd for NFBD (272-345) was 283 +/- 160 nM. Moreover, in comparison with the TCD, NFBD (272-345) formed unstable DNA complexes with a 15-fold faster off-rate. We conclude that the amino acids represented between 266 and 272 termed the complex stabilizing subdomain, influences DNA complex formation independent of DNA binding specificity, and may be one mechanism for heterogeneity of DNA interaction by C/EBP family members.

Amino Acid Sequence↗
PubMed1994

13C-NMR relaxation in three DNA oligonucleotide duplexes: model-free analysis of internal and overall motion.

Natural-abundance 13C-NMR spectra of [d(TCGCG)] (1), [d(CGCGCG)]2 (2), and [d(GGTATACC)]2 (3) were measured at 90.6 MHz to obtain 13C-1H NOEs and T1 relaxation times; relaxation data were also measured at 125.7 MHz for 1 and 2 and at 62.9 MHz for 1. Analysis of the relaxation data was performed in the context of the "model-free" approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559], leading to the following conclusions: (i) Optimized values for the overall correlation times of 0.9 ns for 1 and 1.4 ns for 2 are close to those predicted by light-scattering results on similar molecules [Eimer et al. (1990) Biochemistry 29, 799-811]. (ii) For the nonterminal residues, the "order parameter", S2, is around 0.8 for the protonated base carbons and 0.6 for the sugar carbons, indicating less spatial restriction on the sugar carbons (in the model-free approach, the order parameter is 1 for a rigid body and 0 for a system with completely unrestricted internal motion). (iii) The order parameters for the terminal residues vary over a wide range with the smallest values around 0.2-0.3 for the HO-13C5' and the 13C3'-OH; rational trends are seen in the variation of S2 with chain position in the terminal residues. (iv) The analysis shows that the order parameters are accurate within 15%. (v) The "effective internal correlation time", tau e, is very short for the sugar carbons (30-300 ps) and less well-defined, but probably also short, for the bases. (vi) The analysis indicates that most of the relaxation in DNA is accounted for by S2 and the tau e is so short that a good approximation to any relaxation property, P (e.g., T1, T2, 13C-1H NOE, 1H-1H cross-relaxation rate), is P = S2Prigid, where Prigid is the value for the property in a system without internal motion (the analysis assumes the same isotropic overall motion for both the rigid and flexible bodies).

Base Sequence↗
PubMed1994

8-(4-Bromo-2,3-dioxobutylthio)NAD: a new affinity label for NAD-specific isocitrate dehydrogenase.

8-(4-Bromo-2,3-dioxobutylthio)nicotinamide adenine dinucleotide (8-BDB-TNAD), a new reactive NAD analog, was synthesized by coupling 8-thio-AMP with NMN, followed by condensation with 1,4-dibromobutanedione. Incubation of 160 microM 8-BDB-TNAD with the allosteric pig heart NAD-dependent isocitrate dehydrogenase causes time-dependent inactivation to a limit of 25% residual activity concomitant with incorporation of approximately 1 mol reagent/mol average subunit. In addition to binding sites for NAD and NADH, this enzyme has been shown to have regulatory sites for NADPH and for ADP (R. S. Ehrlich and R. F. Colman 1982, J. Biol. Chem. 257, 4769-4774). Marked protection against enzyme inactivation by 8-BDB-TNAD and incorporation is provided by the regulatory nucleotides NADPH or ADP, while NAD and NADH are less effective. The rate constant for inactivation shows a nonlinear dependence on 8-BDB-TNAD concentration which can be ascribed to reversible formation of an enzyme-reagent complex (KI = 83 microM) prior to an irreversible reaction (kmax = 0.0625 min-1). Analysis of the kinetic properties and binding characteristics of modified enzyme indicates that this enzyme retains the ability to bind ADP, but does not bind NADPH. Thus, 8-BDB-TNAD reacts at or near the allosteric NADPH site of pig heart NAD-dependent isocitrate dehydrogenase.

Affinity Labels↗
PubMed1994

Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation.

The role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation by positive transcription factors was investigated. For this purpose, we constructed a nested set of E. coli rpoD deletion mutants generating carboxy-terminally truncated sigma 70 subunits of RNA polymerase in a high-expression plasmid. The purified mutant sigma 70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity. As expected, since the -35 recognition helix of sigma 70 was deleted in all cases, the mutant enzymes were unable to initiate at factor-independent promoters, except for the special case of perfect "extended minus 10" promoters, at which the need for -35 sequence recognition by RNA polymerase is replaced by recognition of additional base-pairs in the -10 region. However, two factor-dependent promoters, PhoB-dependent PpstS and cAMP receptor protein (CRP)-dependent P1gal, could be activated for transcription by different subsets of the mutant holoenzymes, although these promoters do not contain the perfect extended -10 sequences. These results establish that -35 DNA recognition by sigma 70 is not essential for these cases. Presumably it is replaced by protein-protein contacts between RNA polymerase and the activator, which in both cases is bound to the DNA in a position overlapping the -35 region. Further, the detailed results support the view that the contact and/or activation sites for these two factors may lie on the sigma 70 subunit, within a "contact site II", which extends at least from conserved region 3.2 to the upstream end of region 4.2. Moreover, as in the case of contact site I on the alpha subunit, it appears that contact site II contains various different subsites for interaction with specific class II activators, and that PhoB and CRP require distinct subsites.

Base Sequence↗
PubMed1994

Development of laminar expression of the m2 muscarinic cholinergic receptor gene in rat visual cortex and the effect of monocular visual deprivation.

The postnatal development of laminar pattern of the m2 muscarinic acetylcholine receptor subtype mRNA in the visual cortex of both normally raised and monocularly deprived rats (one eyelid sutured at the age of 11 days) was studied using in situ hybridization histochemistry and computer-assisted image analysis. In normally raised rats, on birth, the m2 transcript was found to be more concentrated in the superficial zones of the cortex. This laminar pattern alters to a more homogeneous distribution of the label throughout the cortex already detectable on day 7. From day 10 onwards a bimodal laminar pattern gradually develops with increased mRNA levels in layer IV and upper layer VI. From postnatal day 21 onwards the hybridization peak in layer VI decreases as compared to the peak level in layer IV resulting in an adult distribution with highest labeling in layer IV, low labeling in layer I to III and moderate labeling in layers V and VI. Monocular deprivation results in decreased m2 mRNA levels in visual cortical layers IV-VI in both deprived and non-deprived cortices already detectable at the age of 18 days and persisting up to the age of 21 days; but this effect disappears following further deprivation until adulthood. The data suggest that the changes in m2 receptor level from a more homogeneous distribution to a bimodal pattern during postnatal development seem to be related to synaptogenesis and final tuning of connectional pattern within the rat visual cortex.

Animals↗
PubMed1994

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: absence of the conserved structural water.

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.

Animals↗
PubMed1994

Assay of the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and the corresponding hydroxyacids by gas chromatography-mass spectrometry.

We developed gas chromatographic-mass spectrometric assays for the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and their corresponding hydroxyacids, lactate, beta-hydroxybutyrate, and beta-hydroxypentanoate (3-hydroxyvalerate) in biological fluids. The corresponding ketoacids, acetoacetate and beta-ketopentanoate, can be assayed simultaneously by pretreating the samples with NaB2H4. The assays involve spiking the samples with deuterated internal standards, deproteinization, ether extraction, and derivatization of the carboxyl groups with (R,S)-2-butanol/HCl and of the hydroxyl groups with chiral (S)-(+)-2-phenylbutyryl chloride. Mass spectrometric analysis is conducted under ammonia positive chemical ionization. We used these assays to follow the metabolism of diol enantiomers in dogs. For (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol, the uptakes from dog plasma of the R and S enantiomer of each diol were identical. In contrast, the metabolism of (S)-1,2-propanediol was faster than that of (R)-1,2-propanediol. (R)-1,2-Propanediol is formed during acetone metabolism, while (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol are potential nutrients. The assays developed will allow further investigations of the metabolisms of acetone, (R)-lactate, and artificial nutrients derived from the 1,3-butanediol and 1,3-pentanediol enantiomers.

3-Hydroxybutyric Acid↗
PubMed1994

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

Adenosylmethionine Decarboxylase↗
PubMed1994

Intravenous dexamethasone pulse therapy in diffuse systemic sclerosis. A randomized placebo-controlled study.

Thirty-five patients with diffuse systemic sclerosis were studied in a randomized, placebo-controlled, double-blind study. Seventeen patients received intravenous dexamethasone "pulse" therapy, while 18 patients received placebo. Each "pulse" consisted of 100 mg dexamethasone in 250 ml 5% dextrose infused intravenously over 1 h. Pulse therapy was repeated every month for 6 months. Assessment of disease status with various parameters was done at entry and at completion of trial, i.e. after 6 months. Significant improvement in skin involvement was seen in the study group, with the total skin score (TSS) decreasing from 28.5 +/- 12.2 to 25.8 +/- 12.8, while in the control group, TSS increased from 30.6 +/- 13.2 to 34.7 +/- 10. Similarly, significant improvement was noted in the flexion index. Other parametres that included extension index, maximum oral opening, range of movement of joints, functional disability score, Raynaud's phenomenon (frequency and duration), ESR, proteinuria, chest X-ray, ECG, lung function tests, barium swallow and antinuclear antibody were unchanged. Adverse effects of therapy were limited to an increased incidence of minor chest infections. It is concluded that intravenous pulse dexamethasone may be useful in the treatment of diffuse systemic sclerosis.

Adult↗
PubMed1994

Liposarcoma of the mesocolon--case report of a rare lesion.

A middle-aged man with a 2-month history of lower abdominal pain was found to have a large left-sided abdominal lump. Radiological investigations (barium enema, ultrasound, and computed tomography scan) revealed a mass lesion in the area of the descending colon, the sigmoid colon, and the rectum. Flexible sigmoidoscopy showed only mucosal edema and luminal narrowing. At laparotomy, a diffuse thickening of both the descending and sigmoid mesocolon extending into the mesorectum was seen, which suggested an inflammatory pathology. A left hemicolectomy with Hartmann's procedure was performed. After obtaining a histopathological diagnosis of liposarcoma of the mesocolon, an abdominoperineal resection of the rectum was done. The patient was advised to undergo postoperative radiotherapy but he did not comply and was thereafter lost to follow-up.

Humans↗
PubMed1994

BamHI restriction fragment length polymorphism (RFLP) at the human GST3 gene locus.

A new restriction fragment length polymorphism (RFLP) detected for the human glutathione S-transferase-pi (GST3) gene with the restriction endonuclease, BamHI (GGATCC) is described. Because of the association of GST isozymes with certain human diseases, the data on involvement of different GST loci, their chromosomal location and information on RFLPs are of potential diagnostic value.

DNA↗
PubMed1994

C3b receptor (CR1) genomic polymorphism in rheumatoid arthritis. Low receptor levels on erythrocytes are an acquired phenomenon.

The number of complement receptor 1 (CR1, CD35) molecules on erythrocytes is genetically determined by two codominant alleles. The numerical expression of CR1 on erythrocytes correlates with a HindIII-RFLP or CR1 gene using CR1-1, a complementary DNA probe. We have found low CR1 on erythrocytes in patients with rheumatoid arthritis (RA) in an Indian population. Low levels in RA patients may be acquired or genetically determined. Fifty-two patients with RA, 48 nonrelated healthy subjects and 19 consanguineous relatives of patients were genotyped. CR1 numbers on erythrocytes were quantitated by the enzyme-linked immunosorbent assay using monoclonal anti-CR1 antibody. Normal subjects and patients were followed up for a period of 6 months to evaluate the stability of their CR1 expression. The gene frequency for allele H and L (7.4- and 6.9-kb HindIII restriction fragment, respectively), which correlated with high and low expression of CR1 on erythrocytes was 0.77 and 0.23 in the normal controls. Gene frequency in RA patients was 0.78 and 0.22 for H and L allele, which did not differ significantly from either controls or relatives (0.80 and 0.20 for H and L allele, respectively). However, RA patients expressed fewer CR1 on erythrocytes within each genotype than their relatives and controls. CR1 on erythrocytes were found to be stable in consecutive samples in controls. In RA patients, the number varied between low and high during the course of the disease. The variation in number was significantly correlated (p < 0.05, r = -0.85 to -0.98) with disease activity as monitored by erythrocyte sedimentation rate. Our results suggest that low levels of CR1 on erythrocytes in patients with RA are not inherited, rather they are acquired during the course of the disease.

Adolescent↗
PubMed1994

The embryology and management of congenital pouch colon associated with anorectal agenesis.

Forty-one infants with a pouch colon malformation accompanied by a high anorectal anomaly were treated between January 1986 and December 1990. The 41 cases constituted 9% of all anorectal malformations and 15.2% of high defects managed during this period. There were 32 boys and nine girls; three of the girls had an associated cloaca. Many of the babies presented in poor condition, with gross abdominal distension caused by the distended colonic pouch. The typical radiological feature was an enormously distended colonic shadow occupying more than 50% of the width of the abdomen. At the time of surgery, the patients were classified into 4 subgroups based on the length of the normal colon. All but three infants had a high wide fistula, with the genitourinary tract consisting of a colovesical fistula in males and a colovaginal or colocloacal fistula in females. Frequent associated malformations included duplication of the appendix and vesicoureteric reflux. The operations performed initially were a window colostomy of the pouch with or without division-ligation of the fistula, end-colostomy after fistula ligation, or subtotal pouch excision with tubularization of the remaining colon and end-colostomy. Thirteen of the 41 patients have undergone a definitive pull-through operation using the posterior sagittal approach, including two children in whom one-stage reconstruction of a cloaca was performed. Standardized management of this complex anomaly is proposed for the initial operation and for definitive reconstruction.

Abnormalities, Multiple↗
PubMed1994

Effect of oxytocin on the concentration of fructose in the accessory glands of mouse.

Studies available in the literature indicate that oxytocin could alter the circulating levels of testosterone and glucose, the prime determinants of fructose synthesis in the male accessory glands. This study was planned to find out if oxytocin could affect the concentration of fructose in the seminal vesicles (SV) and coagulating glands (CG) of mouse. The results show that oxytocin reduces fructose concentration in the SV and CG (SVCG) without altering circulating levels of either glucose or testosterone in the mouse.

Analysis of Variance↗
PubMed1994

Conformation of a T-cell stimulating peptide of interleukin-1 beta protein: circular dichroism studies.

A T-cell stimulating peptide Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the 163-171 fragment epitope of interleukin-1 beta (IL-1 beta), has been synthesized in solution phase and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The backbone conformation of the synthetic fragment, investigated in aqueous solution by circular dichroism (CD) spectroscopy, is qualitatively a mixture of beta-turns and random coil. Quantification of the CD spectra revealed the presence of a 9% beta-turn fraction in water at pH 7.0, suggesting the occurrence of the conformation for the epitope fragment in aqueous solution necessary for T-cell stimulation and antigenicity. Concomitant changes in CD spectra were observed with increases in the trifluoroethanol (TFE) concentration in water, and the beta-turn fraction in peptide increased to 28% at a concentration of 90% TFE. This helicogenic solvent, as well as other solvents such as methanol, acetonitrile and dioxane (all favouring an ordered structure in peptides), failed to induce any alpha-helical conformation in the IL-1 beta (163-171) fragment, and CD spectra were attributed to only beta-turn ordered structure. This beta-turn structure has also been found to be a theoretically preferred conformation using Chou-Fasman proclivity data and is in accordance with the presence of an all-beta-globular conformation for its parent molecule IL-1 beta. Thus, the beta-turn conformation is probably involved in retention of T-cell stimulation activity in this synthetic epitope.

Amino Acid Sequence↗
PubMed1994

Karyotypic findings as an independent prognostic marker in chronic myeloid leukaemia blast crisis.

Fifty-three patients with Ph positive chronic myeloid leukaemia in blastic phase were studied. Additional abnormalities were found in 29 (55%) patients and were more common in myeloid (64%) than lymphoid (45%) blast crisis. The most frequent were +Ph (32%), +8 (28%), +19 (19%), +20 (9%) and +21 (9%). i(17q) (9%) was associated with thrombocytopenia (5/5) and basophilia (2/5). The incidence of additional abnormalities was higher in patients treated with busulphan (70%) than hydroxyurea (44%). No significant differences were noted in the mean values of the clinical and haematological findings recorded at blast crisis between patients with only Ph positive (PP) cells and those with additional abnormalities (AP + AA). Univariate analysis identified karyotypic findings as an independent prognostic marker indicating its significance in assessing the response to therapy and survival after the onset of transformation.

Adult↗
PubMed1994

The free radical system in ischemic heart disease.

The present work was conducted to evaluate the oxygen free radical system in 29 patients and comparing them with nine matched healthy controls of acute and chronic myocardial ischemic syndromes. The parameters assessed for oxidative stress were superoxide anion and malonyldialdehyde, and for the antioxidant defence system were superoxide dismutase, catalase and glutathione reductase. Both oxidative stress and the antioxidant defence system were altered in myocardial ischemia. Subset analysis revealed that in unstable angina and acute myocardial infarction, superoxide anion, malonyldialdehyde and glutathione reductase were elevated while superoxide dismutase and catalase levels were reduced. In stable angina only increased levels of superoxide anion and decreased levels of superoxide dismutase were found. However, this alteration was less marked than in unstable angina and acute myocardial infarction. In the post myocardial infarction group there was no alteration in any of these parameters.

Antioxidants↗