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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 901 records · Page 50Linked to original sources
PubMed1996

Phase combination.

In 1961 Rossmann & Blow published a simple procedure for analytically combining the phase probabilities derived from various isomorphous derivatives or other phase-determining procedures [Rossmann & Blow (1961). Acta Cryst. 14, 641-647]. However, they found it necessary to make an approximation in obtaining the expression for the lack of closure (epsilon) of the phase triangle. In 1970 Hendrickson & Lattman [Hendrickson & Lattman (1970). Acta Cryst. B26, 136-143] suggested an alternative method of defining the lack of closure of the phase triangle which did not require any approximation in deriving the same analytical expression for the phase-probability function. It is now shown that it is possible to avoid the Rossmann-Blow approximation and thereby maintain the original meaning of the lack of closure as defined by Blow & Crick [(1959). Acta Cryst. 12, 794-802] and Dickerson, Kendrew & Strandberg [(1961). Acta Cryst. 14, 1188-1195].

Journal Article↗
PubMed1996

Combination chemotherapy for breast carcinoma using a combination of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) causes a platelet aggregation defect.

Various coagulation parameters including platelet aggregation were evaluated during the course of treatment in 25 patients with breast carcinoma receiving a combination of cyclophosphamide, methotrexate and 5-fluorouracil (CMF). No significant changes were found in whole blood clotting time (WBCT), prothrombin time (PT), kaolin cephalin clotting time (KCCT), platelet count (PC) and prothrombin consumption index (PCI). The CMF combination induced a significant reduction in platelet factor-3 (PF3) availability and reduction in platelet aggregation to ADP and adrenaline. These defects occurred without development of thrombocytopenia. The changes occurred on the 8th day of chemotherapy and a progressive suppression in platelet aggregation was noted during subsequent follow-up. This acquired abnormality in platelet aggregation and PF3 availability may be responsible for various hemorrhagic manifestations such as mucositis, epistaxis and hemorrhagic cystitis following CMF therapy. Further, these changes precede the onset of life-threatening complication of thrombocytopenia. The study of platelet aggregation should be considered besides platelet counting in patients on CMF combination to assess bleeding diathesis.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

dNTP binding site in rat DNA polymerase beta revealed by controlled proteolysis and azido photoprobe cross-linking.

Mild proteolysis of rat DNA polymerase beta (beta-pol) generates an N-terminal 8 kDa domain and a C-terminal 31 kDa domain; the 31 kDa domain is degraded to 6 and 27 kDa fragments by further proteolysis [Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L., & Wilson S.H. (1990) J. Biol. Chem. 265, 2124-2131]. In the present study, we found that more vigorous trypsin digestion of the 27 kDa fragment of beta-pol produces 10 and 12 kDa subdomains. Thus, rat beta-pol has four distinct proteolytic fragments of 8, 6, 10, and 12 kDa, extending from the N-terminus to the C-terminus, respectively. To map the location of the dNTP binding site(s), intact beta-pol was photoaffinity labeled with 8-azido-ATP or 5-azido-dUTP in presence or absence of competitor dNTP (dATP). The labeled enzyme was subjected to controlled proteolysis, and the resulting labeled peptides were separated and sequenced. Competition with dATP showed that three regions of beta-pol in solution combine to form the dNTP binding pocket as follows: residues 4-40 of the 8 kDa domain; residues 142-206 of the 10 kDa subdomain; and residues 263-280 of the 12 kDa subdomain (alpha-helices M and N). These results are discussed in light of the recent crystal structure of dATP bound to rat beta-pol.

Adenosine Triphosphate↗
PubMed1996

Tumor necrosis factor alpha and interleukin 1beta are responsible for in vitro myocardial cell depression induced by human septic shock serum.

Previous studies have demonstrated the presence of myocardial depression in clinical and experimental septic shock. This depression is associated with the presence of a circulating myocardial depressant substance with physical characteristics consistent with cytokines. The present study utilized an in vitro myocardial cell assay to examine the role of various human recombinant cytokines, including tumor necrosis factor (TNF)alpha and interleukin (IL)1beta, in depression of cardiac myocyte contractile function induced by serum from humans with septic shock. The extent and velocity of electrically paced rat cardiac myocytes in tissue culture was quantified by a closed loop video tracking system. Individually, TNF-alpha and IL-1beta each caused significant concentration-dependent depression of maximum extent and peak velocity of myocyte shortening in vitro. In combination, TNF-alpha and IL-1beta induced depression of myocardial cell contractility at substantially lower concentrations consistent with a synergistic effect. Using immunoabsorption, removal of both TNF-alpha and IL-1beta (but not either alone) from the serum of five patients with acute septic shock and marked reversible myocardial depression resulted in elimination of serum myocardial depressant activity. IL-2, -4, -6, -8, -10, and interferon gamma failed to cause significant cardiac myocyte depression over a wide range of concentrations. These data demonstrate that TNF-alpha and IL-1beta cause depression of myocardial cell contraction in vitro and suggest that these two cytokines act synergistically to cause sepsis-associated myocardial depression in humans.

Animals↗
PubMed1996

Clonal T cell expansion induced by interleukin 2 therapy in blood and tumors.

In a phase I clinical trial on the effects of preoperative adjuvant IL-2 therapy given to patients undergoing hepatic resection of colorectal adenocarcinoma metastases, we monitored the putative induction of T cell clonal expansion in both tissues and blood. The presence of T cell clonotypes was analyzed with a PCR-based method that determines V-D-J junction size patterns in T cell receptor (TCR) V beta subfamilies in samples before and after a 5-d IL-2 infusion. This high resolution method analyzing CDR3 sizes of TCR transcripts was used in conjunction with FACS analysis of the corresponding T cell subpopulations with TCR V beta-specific mAb. At time of surgery (day 8 after starting IL-2), we found in the three patients analyzed with V beta-C beta primers multiple dominant T cell clonotypes in the tumor and peritumoral tissues which had probably expanded as a result of therapy. In three control patients not treated with IL-2, multiple oligoclonal patterns were not observed with this set of primers. In the fourth control patient a unique V beta 21-C beta CDR3 pattern which corresponds to two dominant clonotypes was found in the tumor. The same dominant clonotypes identified in the tumor after IL-2 were also detectable in the blood and comparison of the profiles obtained before and after IL-2 therapy indicates that they were induced by IL-2. The relative expansion of the corresponding T cell subpopulations was maintained for varying periods of time after surgery (4-7 d and almost 2 yr in one case). Together, these results indicate that IL-2 induces marked expansion of several T cell clones. Systemic IL-2 administration may represent, either alone or as a vaccine adjuvant, an appropriate way of boosting antigen-specific immune responses.

Adenocarcinoma↗
PubMed1996

The Ty1-copia group retrotransposons in Vicia species: copy number, sequence heterogeneity and chromosomal localisation.

We present an in-depth study of the Ty1-copia group of retrotransposons within the plant genus Vicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploid Vicia species chosen to represent large (V. faba, 1C = 13.3 pg), medium (V. melanops, 1C = 11.5 pg) and small (V. sativa, 1C = 2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, with V. faba containing approximately 10(6) copies, V. melanops about 1000 copies and V. sativa 5000 copies. The degree of sequence heterogeneity of Ty1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons in V.faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.

Amino Acid Sequence↗
PubMed1996

Inhibition of HIV-1 replication in viral mutants with altered TAR RNA stem structures.

Human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans-activation requires the structural integrity of TAR RNA and the cooperative interaction of human host cell proteins. The TAR domain, minimally required for tat response, includes the Tat binding pyrimidine bulge, the TAR RNA upper stem, and the loop sequences. However, little is known about the significance of the 5'-stem structure of TAR in the regulation of viral growth. We designed viral mutations, specifically in the TAR RNA lower stem structure, and studied their effects on the kinetics of viral growth in T-lymphocyte cell lines and in activated human peripheral blood mononuclear cells. Mutations that destabilized the lower TAR stem structure inhibited viral growth to various degrees in different CD4+ T-cells. These results suggest that the structural integrity of the lower stem structure of TAR plays an important role in viral growth, presumably by binding to specific host cell proteins that stabilize Tat-TAR interactions.

Base Sequence↗
PubMed1996

In vivo role of the Sp1 site neighboring sterol-responsive element-1 in controlling low-density lipoprotein receptor gene expression.

The in vivo role of the crucial Sp1 site neighboring sterol-responsive element-1 (SRE-1) in controlling LDL receptor gene expression in the presence or absence of sterols was examined. For this purpose the Xenopus laevis system was utilized as there are two different genes for LDL receptors in frogs which differ in their promoter region in the Sp1-binding sequence of repeat 3 present immediately adjacent to SRE-1. DNase I footprinting of promoters of both receptors showed differences in the affinity of this Sp1 site to purified transcription factor Sp1. Transcript levels of both LDL receptors were measured in livers of frogs on normal and cholesterol-enriched diets. Basal levels and extent of repression of LDL receptor gene on sterol administration were found to be dependent on the nature of the Sp1 site of repeat 3 under in vivo conditions. We conclude that this Sp1 site acts as a constitutive positive transcriptional element that forms a part of the active transcription complex irrespective of cellular sterol levels.

Animals↗
PubMed1996

Granulocytes from chronic myeloid leukemia (CML) patients show differential response to different chemoattractants.

Binding of chemoattractant to polymorphonuclear leukocytes (PMNL) triggers a series of events like polymerization of actin and tubulin, orientation of cells, chemotaxis, increase in fluid pinocytosis and phagocytosis, and stimulation of microbicidal pathways which includes lysosomal degranulation and generation of reactive oxygen species. Earlier studies from our laboratory have shown that stimulation of chemotaxis, fluid pinocytosis, and actin polymerization of CML PMNL in response to a synthetic chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) is significantly lower than that in normal PMNL. It is not known whether this lower response of CML PMNL to fMLP is a global phenomenon involving different chemoattractant receptors or is restricted to the fMLP pathway. We have evaluated chemoattractant induced degranulation process in normal and CML PMNL to fMLP, platelet activating factor (PAF), leukotriene B4 (LTB4), and an analogue of fMLP viz formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenylalanine-O-methionine (FACC8) using release of lysozyme as a parameter. We find that after stimulation with fMLP and FACC8, the mean percent release of lysozyme was significantly lower in CML PMNL as compared to that in normal cells (P < 0.001). There was no significant difference between the two after stimulation with PAF and LTB4. The results indicate that the fMLP pathway is suppressed in CML granulocytes whereas PAF and LTB4 pathways appear unaltered in these cells. We therefore also studied the kinetics of peptide-receptor interaction with a labelled hexapeptide fNLPNTL which binds to the fMLP receptor. Our results show that the number of fMLP receptors/cell is significantly lower in CML PMNL (P < 0.05) than in normal PMNL, while their affinity constants and dissociation constants were comparable.

Chemotactic Factors↗
PubMed1996

Inhibition of plasma-mediated adherence of sickle erythrocytes to microvascular endothelium by conformationally constrained RGD-containing peptides.

Adherence of sickle erythrocytes to vascular endothelium likely initiates or participates in microvascular occlusion, leading to ischemic tissue and organ damage characteristic of sickle-cell pain episodes. In vitro, sickle-cell adherence to endothelium involves adhesive plasma proteins and integrin and nonintegrin receptors on sickle cells and endothelial cells. The involvement of arginine-glycine-aspartic acid (RGD) sequences in adhesive plasma proteins and integrin receptors suggests that RGD-containing peptides may inhibit sickle-cell/endothelial-cell adherence. In the present study, inhibition of plasma-mediated sickle-erythrocyte adherence to endothelium using conformationally constrained RGD-containing peptides was quantified in vitro under continuous flow at a shear stress of 1.0 dyn/cm2. Two conformationally constrained RGD peptides were investigated: 6Z (which has high affinity for alpha5beta1, alpha(V)beta3, and alpha(IIIb)beta3 integrin receptors), and TP9201 (which preferentially binds to alpha(IIb)beta3). Peptide 6Z at 50 microM inhibited plasma-mediated sickle-cell adherence to microvascular endothelium 70% when incubated with sickle red cells, and 63% when incubated with endothelium. Under similar conditions, peptide TP9201 inhibited plasma-mediated sickle-cell adherence up to 85% at concentrations from 250 to 500 microM TP9201. The inhibition of plasma-mediated adherence by conformationally constrained RGD peptides, but not by linear or circular constructs, suggests that the tertiary structure of the peptide containing the binding sequence is important. Inhibition of plasma-mediated sickle-cell adhesion with these peptides in vitro suggests that such conformationally constrained RGD peptides could provide therapeutic interventions in the course of the disease by inhibiting receptor-ligand interactions.

Anemia, Sickle Cell↗
PubMed1996

Tissue and developmental specific expression of murine smooth muscle gamma-actin fusion genes in transgenic mice.

Smooth muscle gamma-actin (SMGA) is an excellent marker of smooth muscle differentiation because it is essentially restricted to smooth muscle. As a first step toward unraveling the mechanisms underlying smooth muscle development and differentiation, we have examined the tissue-specific and developmental expression patterns of six constructs carrying portions of the murine SMGA gene linked to chloramphenicol acetyltransferase (CAT) in stable lines of transgenic mice. Based on the transgenic studies most, if not all, of the regulatory elements necessary for proper spatial and temporal expression of SMGA are present within a 13.7 kb segment of the SMGA gene containing 4.9 kb of upstream sequence, exon 1, intron 1, and a portion of exon 2 up to the start codon for translation. A second construct (SMGA11.6CAT) that lacks the distal 2.1 kb of upstream sequence but is otherwise identical to SMGA13.7CAT shows a similar level of smooth muscle-specific CAT activity. However, SMGA9.3CAT fusion gene containing only 571 bp of 5' flanking sequence, but otherwise identical to SMGA13.7CAT, and SMGA6.0CAT containing only the 4.9 kb upstream sequence, exon 1, and a miniintron 1 show a more than a 100-fold reduction of CAT activity in most smooth muscle-rich tissues. Furthermore, removal of most or all of intron 1 from a transgene with 571 bp of upstream sequence (SMGA2.0 CAT and SMGA0.6CAT) results in a near-complete or complete loss of activity, respectively, in all tissues. Overall, the studies suggest that upstream elements between -2.7 kb and -571 bp and elements within intron 1 are required for high levels of SMGA gene expression in an appropriate temporal-spatial fashion.

Actins↗
PubMed1996

Differential modulation of B7-1 and B7-2 isoform expression on human monocytes by cytokines which influence the development of T helper cell phenotype.

The co-stimulatory molecules B7-1/B7-2 expressed on the surface of antigen-presenting cells have been suggested to influence the development of T helper 1 (Th1)-versus Th2-immune responses. These studies were conducted to elucidate the effect of immunoregulatory cytokines which influence the development of Th1/Th2 immune responses on the expression of the B7 isoforms B7-1 and B7-2 on resting and activated human monocytes and B cells. Interleukin (IL)-4 and IL-10, which induce the development of Th2 immune responses, down-regulated B7-2 and moderately up-regulated B7-1 expression on resting CD14+ monocytes in peripheral blood mononuclear cells. Interferon-gamma (IFN-gamma), which induces the development of Th1 immune responses, enhanced the expression of both B7-1 and B7-2 isoforms. Tumor necrosis factor (TNF)-alpha, which elicits both Th1- and Th2 characteristics depending on experimental conditions, down-regulated B7-2 but did not alter B7-1 expression. The effect of TNF-alpha and B7-2 expression is not mediated through endogenously produced IL-10, as addition of anti-IL-10 antibodies did not restore B7-2 expression. None of the other cytokines tested, including IL-1 alpha, IL-1 beta, IL-2, IL-5, IL-6, IL-12, granulocyte/macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-alpha, modulated the expression of B7 isoforms on resting monocytes. Lipoolysaccharide stimulation of monocytes down-regulated B7-2 and up-regulated B7-1 expression in a manner similar to IL-10. The expression of B7-1 and B7-2 on purified B cells were not altered by any of the cytokines tested, including IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-gamma, TNF-alpha, TGF-alpha and GM-CSF. Taken together, our results suggest that the cytokines which induce Th1/Th2 immune responses exert differential effects on B7 isoform expression on resting monocytes but have no effect on resting or activated B cells.

Adult↗
PubMed1996

Chalcone synthase in rice (Oryza sativa L.): detection of the CHS protein in seedlings and molecular mapping of the chs locus.

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.

Acyltransferases↗
PubMed1996

The Ty1-copia group retrotransposons of Allium cepa are distributed throughout the chromosomes but are enriched in the terminal heterochromatin.

The genomic organization and diversity of the Ty1-copia group retrotransposons has been investigated in a monocotyledonous plant, Allium cepa. We used the polymerase chain reaction (PCR) to generate sequences corresponding to a conserved domain of the reverse transcriptase gene of Ty1-copia retrotransposons in this plant. Sequence analysis of 27 of these PCR products shows that they are a highly heterogeneous population, a feature which is common in plants but not in yeast and Drosophila. Slot-blot analysis shows there are 100,000-200,000 copies of Ty1-copia group retrotransposons within the A. cepa genome (2C = 31.7 pg), indicating that they are a significant component of the genome of this plant. In situ hybridization to metaphase chromosomes reveals that Ty1-copia retrotransposons are distributed throughout the euchromatin of all chromosomes of A. cepa but are enriched in the terminal heterochromatic regions, which contain tandem arrays of satellite sequences. This is the first clear evidence for the presence of Ty1-copia retrotransposons in the terminal heterochromatin of plants and contrasts with the distribution of these elements in other plant species.

Allium↗
PubMed1996

An assessment of nonspecific adsorption to Eudragit S-100 during affinity precipitation.

The problem of nonspecific adsorption to the reversibly soluble-insoluble polymers is of considerable importance in the design of an affinity precipitation protocol. It was seen that activation and coupling of the affinity ligand to the polymer changes the nature of the polymer surface in a significant fashion. The results with pure trypsin, partially purified trypsin preparation, and crude protein extract-containing protein inhibitors of trypsin and alpha-amylase and the reversibly soluble-insoluble polymer Eudragit S-100, show that nonspecific adsorption may not be a severe limitation in such systems.

Acrylic Resins↗