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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 991 records · Page 55Linked to original sources
PubMed1995

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, X-ray, and NMR.

The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

Amino Acid Sequence↗
PubMed1995

The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats.

The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding beta-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G beta-homologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the beta-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of beta-transducin homologs. Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of beta-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.

Amino Acid Sequence↗
PubMed1995

Dicationic diarylfurans as anti-Pneumocystis carinii agents.

Seven dicationic 2,5-diarylfurans have been synthesized, and their interactions with poly(dA-dT) and the duplex oligomer d(CGCCAATTCGCG)2 were evaluated by Tm measurements. The inhibition of topoisomerase II isolated from Giardia lamblia, the inhibition of growth of G. lamblia in cell culture by these furans, and the effectiveness of these compounds against Pneumocystis carinii in the immunosuppressed rat model have been assessed. Strong binding affinities to poly(dA-dT) and to the oligomer were observed for the dicationic furans, and the interaction strength is directly correlated to the biological activity of the compounds. An X-ray structure for the complex of the dicationic amidine derivative, 2,5-bis(4-guanylphenyl)furan (1), with the oligomer demonstrates the snug fit of these compounds with the AATT minor-groove binding site and hydrogen bonds to AT base pairs at the floor of the minor groove. The stronger DNA binding molecules are the most effective inhibitors of topoisomerase II and G. lamblia in cell culture, and there is a correlation for both DNA interaction and topoisomerase II inhibition with the biological activity of these compounds against G. lamblia. Compound 1 is the most effective against P. carinii, it is more active and less toxic than pentamidine on intravenous administration and it is also effective by oral dosage. The results presented here suggest a model for the biological action of these compounds in which the dication first binds in the minor groove of DNA and forms a complex that results in the inhibition of the microbial topoisomerase II enzyme.

Animals↗
PubMed1995

A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli.

Most of the sigma (transcriptional initiation specificity) subunits of RNA polymerase, from a wide range of eubacteria, show strong elements of amino acid sequence similarity. There is evidence that two of the "conserved" regions, 2.4 and 4.2, are involved in recognition of the consensus DNA sequences centred near -10 and -35, respectively, which define promoter sites for the initiation of transcription. Since all the alternative sigma subunits of the above type function by binding to a common core polymerase enzyme in a given bacterium, it can be predicted that a hybrid sigma might be functional, and if so should permit RNA polymerase to initiate only at a correspondingly hybrid promoter. To test these predictions, a hybrid gene encoding the amino-proximal 529 amino acids of the major Escherichia coli sigma protein, sigma 70 (including region 2.4) followed by the last 82 amino acids of the heat-shock sigma protein, sigma 32 (including region 4.2) was constructed and fused to Plac on a plasmid. Major-consensus, heat-shock and hybrid promoters were fused to a chloramphenicol acetyl transferase (CAT) reporter gene on a compatible plasmid. CAT assays showed that, as predicted, a promoter with a "heat-shock" -35 consensus and a "major" -10 consensus sequence (PHM) required Plac-dependent production of the hybrid sigma (sigma 70-32) for activity in vivo. PHM then became a strong promoter. The hybrid sigma gene has potential advantages over its parents for structure-function studies.

Amino Acid Sequence↗
PubMed1995

In vivo local expansion of clonal T cell subpopulations in renal cell carcinoma.

Renal cell carcinoma (RCC) is one human tumor to which the immune response may control the growth of tumor cells. These tumors are infiltrated by a large mononuclear infiltrate mainly composed of T lymphocytes. To characterize the lymphocytes infiltrating RCC, we analyzed the molecular structure of the T cell receptor (TCR) alpha and beta chains in tumor and paired peripheral blood lymphocytes from a series of 6 untreated patients. We first determined V alpha and V beta gene segment usage by PCR using a panel of V specific oligonucleotide primers (V alpha 1-w29 and V beta 1-w24). A highly diverse usage of TCR V alpha and V beta gene usage was observed in 5 of 6 tumors. In addition, the few tumor overexpressed V beta specificities detected by reverse transcription-PCR were shown to contain minor T cell expansions. Strikingly, 1 of the 6 tumor studied displayed a skewed TCR repertoire with V beta 4 transcript representing 25% of the TCR signals. Clonality of the tumor overexpressed V beta transcripts was analyzed by CDR3 size distribution analysis. In the particular tumor displaying a biased repertoire large expansions of T cell subpopulations were detected (particularly in V beta 4) specifically at the tumor site. Such T cells may be expanded locally in response to tumor antigens.

Adult↗
PubMed1995

Sleep, its subjective perception, and daytime performance in insomniacs with a pattern of alpha sleep.

Intrusion of alpha activity, an electroencephalographic (EEG) pattern typical for wakefulness, into sleep stages has repeatedly been described and investigated in various populations. Some studies suggested that it is a less deep and restorative sleep, but others did not support this interpretation. The present study was carried out to collect ample data on neurophysiology and subjective experience of sleep and on daytime cognitive performance to clarify this point. A sample of 128 primary insomniacs was investigated with polysomnography (PSG) that was submitted to a computerized, automatic analysis of alpha activity during sleep. It yielded two groups of 64 Ss each with a normal, that is, nonalpha sleep EEG and with alpha-sleep, respectively. Contrasting the two groups for PSG showed that alpha sleep Ss had significantly more stage 4 and a (nonsignificant) tendency for more awakenings. Subjectively, they largely underestimated intermittent wake time and consequently overestimated sleep duration by 50 min. The performance test battery showed a difference in one test only, that is, a better short-term memory function by alpha sleep Ss. In conclusion, there was no result supporting the assumption that alpha sleep is less restorative, but a significant lack of perception of intermittent awakenings during night sleep by alpha sleep Ss was found. The authors propose an explanation and point to the implications this misperception might have for the clinician.

Alpha Rhythm↗
PubMed1995

Coronary angioplasty for the control of intractable ventricular arrhythmia.

Ventricular arrhythmias (VAs) that occur following an acute extensive anterior myocardial infarction (MI) usually respond to conventional antiarrhythmic regimes of treatment. Rarely, the VA may prove intractable to therapy. This report is of three patients who presented at varying time frames (3 h to 10 weeks) following an anterior MI. They exhibited sustained monomorphic ventricular tachycardia and hemodynamic instability despite multiple antiarrhythmic drug therapy, intravenous magnesium, direct-current cardioversion (DCCV), overdrive pacing (in one case), and intra-aortic balloon counterpulsation (IABP). Although there was no clinical evidence of continuing ischemia and although coronary angiography that was done in each case showed the infarct-related artery (IRA) to subtend akinetic areas on left ventricular (LV) angiogram, percutaneous transluminal coronary angioplasty (PTCA) of the IRA was done in all three cases. Reestablishing patency of the IRA helped in controlling the VA dramatically with average therapeutic doses of antiarrhythmic drugs. All three patients showed this control to have been maintained over a follow-up period of more than 1 year, with partial improvement in LV function and signal-averaged electrocardiogram negative for late potentials. Thus, in patients with extensive infarction and intractable VA, PTCA of the IRA may provide control of VA even in the absence of clinical signs of active ischemia or viable muscle mass.

Angioplasty, Balloon, Coronary↗
PubMed1995

Serotonin and serotonin antagonism in cardiovascular and non-cardiovascular disease.

Serotonin, or 5-hydroxytryptamine, is a naturally-occurring vasoactive substance found primarily in the brain, enterochromaffin tissue, and blood platelets. It has diffuse cardiophysiologic effects. The multiple effects of serotonin on blood vessels can be explained by the existence of 2 serotonergic receptor subtypes (the S1 receptor mediates vasodilation, and the S2 receptor vasoconstriction). Serotonin via the S2 receptor also augments the actions of several other vasoconstricting substances. Serotonin may be responsible for causing, or at least perpetuating, some forms of systemic hypertension through peripheral and central nervous system (CNS) actions. Ketanserin is a highly selective S2-serotonergic antagonist with additional alpha-adrenergic blocking activity, which has been proposed as a therapy for various cardiovascular diseases including hypertension. It has been shown to be more effective than placebo in treating hypertension and comparable in effectiveness to other antihypertensive drugs. Its major side effects relate to the CNS, and prolongation of the electrocardiogram QT interval has been described. Caution must be used when using ketanserin in patients receiving potassium- and magnesium-losing agents, because of the risk of torsades de pointes. Ketanserin has potential utility in the treatment of eclampsia, peripheral vascular disease, carcinoid syndrome, and "shock lung." The drug is not yet approved for clinical use in the United States.

Animals↗
PubMed1995

Endothelin and endothelin antagonism: roles in cardiovascular health and disease.

Endothelin is the most potent mammalian vasoconstrictor yet discovered. Its three isoforms play leading roles in regulating vascular tone and causing mitogenesis. The isoforms bind to two major receptor subtypes (ETA and ETB), which mediate a wide variety of physiologic actions in several organ systems. Endothelin may also be a disease marker or an etiologic factor in ischemic heart disease, atherosclerosis, congestive heart failure, renal failure, myocardial and vascular wall hypertrophy, systemic hypertension, pulmonary hypertension, and subarachnoid hemorrhage. Specific and nonspecific receptor antagonists and ECE inhibitors that have been developed interfere with endothelin's function. Many available cardiovascular therapeutic agents, such as angiotensin-converting-enzyme inhibitors, calcium-entry blocking drugs, and nitroglycerin, also may interfere with endothelin release or may modify its activity. The endothelin antagonists have great potential as agents for use in the treatment of a wide spectrum of disease entities and as biologic probes for understanding the actions of endothelin in human beings.

Amino Acid Sequence↗
PubMed1995

Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines.

Thermal stress induces expression of a family of heat shock proteins which may regulate the synthesis of various cellular genes. We investigated the effect of heat shock on polyadenylation in Epstein-Barr Virus (EBV) negative and EBV transformed human Burkitt's lymphoma (BL) B-cell lines. Incubation of the BL B-cell line P3HR-1, carrying the defective EBV genome [EBV nuclear antigen-2 gene deletion] at 46 degrees C for 15 min increased nuclear poly(A) polymerase (PAP) activity. Thereafter, enzymatic activity declined and at 60 min it was reduced to about 50% of that observed in cells incubated at 37 degrees C. In contrast, no significant increase in PAP activity was observed at 15 min or thereafter in an EBV- BL cell line, ST-486, in response to elevated temperature. Furthermore, no heat shock mediated change in nuclear poly(A)-specific endonuclease activity was observed in either P3HR-1 or ST-486 cells suggesting a specific effect on PAP activity. However, thermal stress dependent increase in c-myc expression was detected only in P3HR-I cells. These results suggest an association between EBV transformation and enhanced expression of c-myc and PAP activity. To further determine the role of EBV, and EBV- BL cell line, BL-30, and BL-30 cells infected in vitro with a wild type strain of EBV, BL-30/B95-8, were investigated. BL-30/B-95-8, unlike the parental BL-30 cells, exhibited c-myc and PAP gene upregulation at 15 min but were downregulated at 60 min following exposure of cells to elevated temperatures. These results suggest that infection of human B-cells with EBV is associated with their ability to respond to thermal stress by increased PAP activity which may stabilize mRNA through enhanced polyadenylation.

Burkitt Lymphoma↗
PubMed1995

Long-term culture of primary B cells and in vitro expression of an exogenous gene.

Efficient introduction and expression of exogenous genes into primary B cells is very important to study B-cell biology and is essential for gene therapy. These efforts have often been impeded by the lack of availability of a simple culture condition for growth and proliferation of primary B cells as well as the lack of vehicles for efficient introduction of genes of interest. In this communication, we have developed a culture condition that supports the growth of primary B cells from beta-gal-immunized mouse spleen for 30 days without the aid of feeder layers. During this period, B cells secreted polyclonal antibodies into the medium. To study expression of an exogenous reporter gene, human growth hormone (hGH) was introduced into cultured B cells using retroviral vectors. hGH was expressed up to 21 days in the absence of drug selection and the infected cells continued to secrete immunoglobulins into the medium.

Animals↗
PubMed1995

Effect of cisplatin and FK565 on the activation of tumor-associated and bone marrow-derived macrophages by Dalton's lymphoma.

The present investigations were undertaken to study the effect of Dalton's lymphoma (DL) in situ on the functions of DL-associated macrophages (DL-AM) and bone marrow-derived macrophages (BMDM) in C3H/He mice. DL-AM showed enhanced production of reactive nitrogen intermediates (RNI) and tumor necrosis factor (TNF) compared with normal peritoneal macrophages (NMO). BMDM of DL mice also showed enhanced production of RNI compared with BMDM of normal mice. These observations suggest that the presence of DL in situ creates an environment which favours the activation of both DL-AM and macrophage progenitors located at a distant site. The effect of in vivo administration of chemotherapeutic drugs cisplatin and FK565 on the activation of DL-AM by DL cells was also investigated. Both cisplatin and FK565 augmented RNI production of NMO but differed in their effect on DL-AM. The production of RNI by DL-AM of cisplatin-treated mice was inhibited, whereas in the FK565 group it was up-regulated.

Animals↗