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J Kuchibhotla

Publications and source records attributed to J Kuchibhotla.

16 recordsLinked to original sources
PubMed1998

Nuclear factor kappaB dominant negative genetic constructs inhibit X-ray induction of cell adhesion molecules in the vascular endothelium.

X-ray-induced expression of inflammatory mediators has been proposed to contribute to radiation injury in normal tissues. Radiation-inducible inflammatory mediators include the cell adhesion molecule (CAM) E-selectin and the intercellular adhesion molecule (ICAM)-1. Nuclear factor (NF)kappaB is activated by X-rays and may participate in the transcriptional regulation of each of these inflammatory mediators. To determine whether NFkappaB inhibition abrogates X-ray induction of inflammatory mediators, we used two experimental approaches including NFkappaB inhibitory drugs and a dominant negative genetic construct. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells were treated with the NFkappaB inhibitors ALLN, PDTC, NAC, and MG132. After irradiation, E-selectin or ICAM-1 was measured by fluorescence-activated cell-sorting analysis. E-selectin and ICAM-1 expression was measured by use of immunofluorescence and fluorescence-activated cell-sorting analysis. E-selectin expression increased 7-fold, and ICAM-1 expression increased 4-fold after irradiation. All of the inhibitors attenuated E-selectin expression after irradiation. ALLN and MG132 attenuated radiation-induced ICAM expression. However, PDTC and NAC induced increased expression of ICAM-1 in HUVECs. Inhibition of X-ray induction of ICAM by these agents could not be demonstrated. In separate experiments, the NFkappaB dominant negative genetic construct was cotransfected with the promoter-reporter constructs by means of Lipofectin reagent. The ICAM promoter-reporter construct consists of the 1.2-kb segment of the human ICAM promoter upstream of the transcriptional start site linked to the luciferase reporter gene (pGL.FL-Luc). The E-selectin promoter-reporter construct consists of 525 bp upstream of the transcriptional start site of the human E-selectin promoter linked to the human growth hormone reporter gene (pE525-GH). Endothelial cells transfected with the ICAM-1 promoter-reporter construct showed a 3-fold induction after irradiation. Likewise, cells transfected with pE525-GH showed a 7-fold induction after irradiation. When cotransfected with the CAM reporter-promoter constructs, the NFkappaB dominant negative genetic construct abolished X-ray-induced transcriptional activation of the E-selectin and ICAM-1 promoters. NFkappaB inhibition is, therefore, a means of abrogating radiation-induced expression of CAMs.

Cell Adhesion↗
PubMed1997

TNF-alpha and IL-1 upregulate membrane-bound and soluble E-selectin through a common pathway.

BACKGROUND: Endothelial cell adhesion molecules such as E-selectin promote the capture of neutrophils (PMN) in the microcirculation and initiate the inflammatory response. In contrast, when "shed" into the microcirculation, soluble E-selectin can bind PMN in the blood stream, reducing the number available for adhesion to injured tissue. These experiments were designed to better characterize the molecular response to cytokines and the balance between cell surface (bound) and soluble (unbound) E-selectin. METHODS: Cultured human umbilical veins, exposed to human recombinant TNF-alpha or IL-1 (10 pg/ml), were analyzed for E-selectin mRNA induction (Northern blot), E-selectin cell surface expression (flow cytometry), and sE-selectin release (ELISA). Transcriptional regulation was analyzed via Raf kinase dominant negative gene transfection. RESULTS: E-selectin mRNA expression was markedly increased at 2 h and sustained through 8 h. No further induction was noted at 12 h. Upregulation of cell surface E-selectin was noted (mean fluorescence) as early as 2 h for TNF-alpha (baseline, 12.28 +/- 1.32; TNF-alpha, 23.03 +/- 1.81) or 4 h for IL-1 (baseline, 12.28 +/- 1.32; IL-1, 70.00 +/- 3.04) with maximum expression at 6 h (TNF-alpha, 118.8+/-15; IL-1, 94.11 +/- 9. 34). Expression returned to baseline levels by 24 h. Soluble E-selectin (ng/ml) assays demonstrated later increases beginning at 12 h (TNF-alpha, 0.313 +/- 0.077; IL-1, 0.159 +/- 0.075) and continuing through 24 h (TNF-alpha, 0.340 +/- 0.062; IL-1, 0.157 +/- 0.030). Transfection of endothelial cells with Raf kinase 301 dominant negative gene resulted in proportionate decreases in the peak expression in both surface (bound) E-selectin (TNF-alpha, 51. 7%; IL-1, 29.6%) and sE-selectin (TNF-alpha, 49.2%; IL-1, 34.5%). CONCLUSION: The temporal sequence of late decreases in cell surface E-selectin accompanied by increases in soluble E-selectin indicates that the source of E-selectin in the microcirculation is shed receptors rather than synthesis of a different type of receptor. Enhancement of such "shedding" may decrease PMN adhesion to injured tissue and have therapeutic potential.

Cell Adhesion↗
PubMed1997

Sialyl Lewis X mimetics attenuate E-selectin-mediated adhesion of leukocytes to irradiated human endothelial cells.

Ionizing radiation causes histological changes in normal tissues that resemble those resulting from the inflammatory response. Inflammation is a multistep process requiring expression of adhesion molecules on the surface of endothelial cells which results in leukocyte extravasation. E-selectin is an adhesion molecule that mediates leukocyte "rolling" on the endothelium and is required for the inflammatory response. We quantified E-selectin expression and selectin-dependent adhesion of leukocytes to human endothelial cells after X irradiation to determine whether E-selectin participates in the radiation-mediated inflammation-like response. Immunofluorescence staining of irradiated endothelial cells demonstrated expression of E-selectin on the cell surface similar to that elicited by treatment with interleukin-1 (IL-1). Radiation-mediated expression of E-selectin was dependent on dose and time and occurred at doses as low as 0.5 Gy. Furthermore, the increased adhesion of leukocytes to irradiated endothelial cells was prevented by an E-selectin-blocking antibody. Sialyl Lewis X is one of the molecules on the surface of leukocytes that adheres to E-selectin. The anti-inflammatory agents glycyrrhizin and carminic acid, which are structural analogues of sialyl Lewis X, attenuated adhesion of leukocytes to endothelial cells treated with X rays or IL-1. These data implicate a new class of anti-inflammatory agents in the prevention of adhesions of leukocytes to the irradiated vascular endothelium.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Cell adhesion molecules mediate radiation-induced leukocyte adhesion to the vascular endothelium.

The predominant early histological changes in irradiated tissues are edema and leukocyte infiltration. Cell adhesion molecules (CAMs) are required for the extravasation of leukocytes from the circulation. To study the role of CAMs in the pathogenesis of radiation-mediated inflammation, we quantified the expression of P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 glycoproteins on the surface of irradiated human endothelial cells. We found that E-selectin and ICAM-1 expression increased after irradiation, whereas there was no increased expression of other cytokine-inducible adhesion molecules (P-selectin or vascular cell adhesion molecule-1). We found a dose- and time-dependent increase in radiation-induced expression of both E-selectin and ICAM-1. Furthermore, the threshold dose for E-selectin expression was 1 Gy, whereas the threshold dose for ICAM-1 synthesis was 5 Gy of X-rays. Northern blot analysis of RNA from irradiated endothelial cells demonstrated that ICAM-1 is expressed at 3-6 h following irradiation. No de novo protein synthesis was required for increased ICAM-1 mRNA expression. The 1.1-kb segment of the 5' untranslated region of the ICAM-1 gene was sufficient for X-ray induction of chloramphenicol acetyltransferase reporter gene expression. We measured whether ICAM-1 mediates adhesion of leukocyte to the irradiated endothelium and found that leukocyte adhesion occurred concurrently with ICAM-1 induction. Radiation-mediated leukocyte adhesion was prevented by anti-ICAM-1 blocking antibodies. These data indicate that ICAM-1 participates in the inflammatory response to ionizing radiation. Moreover, radiation induction of these CAMs occurs in the absence of tumor necrosis factor and interleukin 1 production.

Antibodies↗
PubMed1996

Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts.

PURPOSE: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. METHODS AND MATERIALS: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1 and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. RESULTS: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. CONCLUSIONS: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

Ataxia Telangiectasia↗
PubMed1995

E-selectin gene induction by ionizing radiation is independent of cytokine induction.

The mechanism of the x-ray-mediated inflammatory response in normal tissues is unknown. To determine whether leukocyte infiltration into irradiated tissue is regulated by adhesion molecule expression, we quantified the synthesis of glycoproteins that participate in inflammation. We found that E-selectin is synthesized in a time-dependent manner following exposure to doses as low as 0.5 Gy. Northern blot analysis demonstrated that E-selectin mRNA expression increased at 2 h after x-irradiation and increased expression required no de novo protein synthesis. Transcription of the promoter region of E-selectin (-578 to +35) was transiently induced following x-irradiation, whereas deletion of the NFkB binding site eliminated x-ray induction. Electrophoretic mobility gel shift analysis confirmed increased binding of nuclear proteins from irradiated endothelial cells to the NFkB binding sequence from the E-selectin promoter. Nuclear protein binding to the NFkB binding sequence was altered by antibodies to the p50 and p65 components of NFkB. These data demonstrate that E-selectin expression does not require cytokine synthesis, but involves NFkB activation.

Base Sequence↗
PubMed1994

Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.

Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production.

Amino Acid Sequence↗
PubMed1993

Radiation signaling mediated by Jun activation following dissociation from a cell type-specific repressor.

The promoter regions of several radiation-inducible genes contain AP-1 cis-acting regulatory elements that are dependent upon protein kinase C signaling. We analyzed nuclear protein from irradiated human tumor cell lines for binding to the AP-1 consensus sequence. The increase in nuclear protein binding following irradiation was specific for the AP-1 sequence and was reduced by antibodies to c-Jun and c-Fos. The AP-1 DNA binding sequence was found to regulate transcription in irradiated cells and mutation of the AP-1 site within the c-jun promoter abolished transcriptional induction by radiation. The gene encoding the chimeric transcription factor Gal4-Jun5-253, which includes the DNA binding region of Gal4 and the transcriptional regulatory region of c-Jun, was cotransfected with the reporter plasmid with Gal4 binding sequences (G5B-CAT). Transfection of RIT-3 and HeLa cells revealed that the regulatory region of Jun was sufficient to activate transcription following irradiation. Conversely, Hep G2 cells, which do not contain the cell type-specific Jun repressor, were not responsive to radiation-induced Jun activation. The c-Jun repressor was found to regulate Jun activation by experiments using the expression vector CMV-jun, which competes for Jun inhibitor and eliminates radiation-induction of Jun. We propose transcription factor dissociation from inhibitor proteins may participate in the initiation of cellular responses to ionizing radiation.

Chloramphenicol O-Acetyltransferase↗
PubMed1985

Generation of monoclonal antibodies recognizing neuronal elements in formalin-fixed paraffin-embedded human tissue.

We used formalin-fixed human spinal cord and dorsal root ganglia as immunogens to generate monoclonal antibodies (mAb) which immunohistochemically react with neurons in formalin-fixed human tissue sections. Three of the mAb recognized all neuronal populations studied, including those in spinal cord, dorsal root ganglia, cerebellum, and cerebrum. A fourth mAb recognized neurons within spinal cord, dorsal root ganglia and dentate nucleus of cerebellum but not those in cerebrum or cerebellar hemispheres. This mAb, unlike the other three, did not recognize murine neurons. These data indicate the feasibility of generating mAb suitable for analysis of human pathological material in its most readily available form, formalin-fixed paraffin-embedded tissue.

Antibodies, Monoclonal↗
PubMed1983

High-molecular weight kininogen. A secreted platelet protein.

Human platelets were studied immunochemically to determine if they contain high-molecular weight kininogen. On crossed immunoelectrophoresis with total kininogen antisera (antisera that recognizes both high- and low-molecular weight kininogen) extracts of platelets contained total kininogen antigen. Platelet total kininogen antigen showed complete antigenic identity with plasma total kininogen and displayed the same electrophoretic migration as plasma total kininogen. Using antisera monospecific to high molecular weight kininogen, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to directly measure platelet high-molecular weight kininogen. By CELISA, 27-101 ng of high molecular weight kininogen antigen per 10(8) platelets was quantitated in detergent-soluble lysates of washed human platelets from nine normal donors with a mean level of 60 ng +/- 24/10(8) platelets. Plasma high-molecular weight kininogen, either in the platelet suspending medium or on the surface of the platelets, could only account for 5% of antigen measured in the solubilized platelets. On the CELISA, platelet high-molecular weight kininogen was immunochemically identical to plasma and purified high-molecular weight kininogen. Platelet high-molecular weight kininogen was secreted from platelets after exposure to ionophore A23187 (3-15 microM), collagen (5-150 micrograms/ml), and thrombin (1.6 U/ml). Secreted platelet high-molecular weight kininogen did not become a part of the platelet Triton-insoluble cytoskeleton. On cross immunoelectrophoresis secreted platelet total kininogen antigen had a similar electrophoretic migration to plasma total kininogen. Thus, human platelets contain high-molecular weight kininogen that can be secreted from platelets and that may participate in plasma coagulation reactions.

Adult↗
PubMed1977

Phase separation in phosphatidylcholine bilayers as a predictor of inhibition of blood platelet aggregation by amantadines.

The ability of eleven amantadine derivatives to induce phase separation in dipalmitoyl phosphatidylcholine bilayers was studied by differential scanning calorimetry. The relative potency varied with the shape and size of the hydrocarbon cage. These agents also markedly inhibited blood platelet aggregation. The relative potencies of these compounds to induce phase separation showed a significant correlation (r = 0.70) with their platelet inhibitory activity suggesting that their pharmacologic action may be at the level of the platelet membrane. The effective concentration of the parent component amantadine is similar to its pharmacologic concentration suggesting its use as an anti-platelet drug.

Amantadine↗
PubMed1976

Potassium uptake and release by human blood platelets.

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ -free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first-order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta-glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01-0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

Aspirin↗