Biomedical subjects

D K Agrawal

Publications and source records attributed to D K Agrawal.

At least 55 records · Page 3Linked to original sources

PubMed1995

Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: possible involvement of tyrosine kinase and protein kinase C.

Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37 degrees C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa alpha-subunit of the inhibitory G-protein was identified by specific antibodies to Gi alpha-1/2 and Gi alpha-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa Gi alpha subunit was increased 3.4 fold in differentiated cells. The expression of a 43 KDa Gs alpha subunit identified by Western blotting using specific antibody to Gs alpha and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa Gs alpha subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [35S]GTP gamma S binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of Gi alpha-1/2 and Gs alpha subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of Gi alpha-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.

Alkaloids↗

PubMed1995

Assembly of the T = 4 Nudaurelia capensis omega virus capsid protein, post-translational cleavage, and specific encapsidation of its mRNA in a baculovirus expression system.

We have expressed the gene encoding the coat protein (CP) of Nudaurelia capensis omega virus in insect cells with a baculovirus vector. Expression of CP resulted in formation of virus-like particles (VLPs) having a size consistent with the T = 4 quasi-symmetry observed for native virions. This is the first demonstration of assembly for a T = 4 particle, with chemically identical subunits present in four distinct environments, by heterologous expression. Initial yields of VLPs were low, and an efficient one step nondenaturing procedure involving separation on discontinuous glycerol gradients was developed. Using this method, VLPs were obtained in quantities sufficient for further characterization. Electron microscopic observation revealed 40-nm particles that were morphologically similar to native virus. SDS-PAGE revealed that these particles were composed of a 62-kDa major protein and a minor 70-kDa protein. Pulse-chase experiments revealed that the larger species was processed into the smaller one very slowly over the course of an infection. It was also determined that this cleavage was apparently dependent on release of these particles from the cell. Furthermore, these particles were found to encapsidate the polyhedrin promoter-directed CP mRNA with an apparently striking degree of specificity and selectivity. This investigation establishes that a specific encapsidation signal exists within the CP coding sequences and that the components required for reconstructing most, if not all, steps in the morphogenesis of this virus can be accomplished in baculovirus-infected cells. The results presented here are consistent with the belief that similar biological strategies are utilized by T = 3 nodaviruses and T = 4 tetraviruses in particle assembly.

Animals↗

PubMed1995

Arterial embolism from anatomical variation at the thoracic outlet.

Thoracic outlet syndrome (TOS) represents a constellation of symptoms arising from the compression of the neurovascular bundle as it exits the thorax. We report a unique case of multiple rare anatomical anomalies resulting in TOS manifested by distal arterial embolism. These anomalies include the combination of: (1) a unilateral right cervical rib, Gruber's type II (Gruber 1869), (2) nonunion of the first thoracic rib, (3) abnormal fibrous insertions of the anterior scalene muscle onto the epineurium of the brachial plexus and adventitia of the subclavian artery, (4) anterior position of the brachial plexus in relation to the third portion of the subclavian artery, and (5) the bifurcation of a single root of the phrenic nerve at the level of the anterior scalene muscle. This series of findings suggest an underlying developmental abnormality with a delayed onset of symptomatology consisting of the thoracic outlet syndrome.

Abnormalities, Multiple↗

PubMed1995

University of Wisconsin solution effects on intimal proliferation in canine autogenous vein grafts.

This study was carried out to investigate if the University of Wisconsin solution (UWs) is suitable for long-term preservation of autogenous vein grafts (AVG) prior to transplantation, compared to autologous whole blood (AWB) and normal saline (NS). Autogenous jugular and femoral veins were harvested from adult mongrel dogs, using a "no-touch" technique. One segment of vein was immediately implanted to serve as a control, while other segments were stored for 24 hr at 4 degrees C in AWB, NS, or UWs. The control and the preserved veins were implanted as reversed interposition grafts in the common carotid or femoral artery positions. After 6 weeks, scanning electron microscopy revealed an intact endothelial cell monolayer in all vein grafts that was stained positively for factor VIII. The degree of intimal thickening, as assessed by light microscopy in the middle position of the grafts, was similar in controls (52.0 +/- 15.8 microns) and in veins stored in UWs (58.5 +/- 16.1 microns), but it was significantly increased (P < 0.05) in veins preserved in NS (198.9 +/- 19.5 microns) and in AWB (312.0 +/- 171.6 microns). Isometric tension studies revealed that maximum contraction and sensitivities (assessed by EC50) to norepinephrine were significantly reduced (P < 0.05) in AVG stored in AWB (0.05 +/- 0.02 g/mm2 and 5.5 +/- 2.8 microM), but not in UWs (0.16 +/- 0.03 g/mm2 0.92 +/- 0.34 microM) and NS (0.09 +/- 0.03 g/mm2 and 3.0 +/- 1.1 microM), compared with controls (0.17 +/- 0.03 g/mm2 and 0.99 +/- 0.38 microM). Acetylcholine (Ach)-induced maximum relaxations were similar in all of the veins.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1995

Effect of endothelin-1 on arterial response to Bay K 8644. A comparison of ischemic and nonischemic arteries.

Endothelin-1 (ET-1) has been proposed as one of the possible mediators of the vasoconstriction seen following ischemia and reperfusion. We investigated the effect of ischemia and reperfusion on the contractile response of canine renal and iliac arteries to the dihydropyridine-type calcium channel agonist (+/-)Bay K 8644, following subthreshold doses of ET-1. No significant difference in the maximum tension was observed between the ischemic and nonischemic arteries in response to Bay K 8644 in the absence of ET-1. The addition of subthreshold dose of ET-1 (10(-10) M) resulted in a significant increase in sensitivity to Bay K 8644 in both the ischemic-reperfused and non-ischemic-reperfused arteries, with a 38 fold increase in iliac arteries and about 8 fold increase in the renal arteries. However, the ET-1 potentiated response was enhanced in the ischemic-reperfused in comparison to the non-ischemic-reperfused vessels in the iliac artery. These data suggest that the potentiating mechanism of ET-1 is not only intact, but enhanced in ischemic-reperfused vessels. Since the enhanced release of ET-1 in vivo is preceded by ischemia and reperfusion, the vasospastic phenomenon observed following these events could well be mediated by ET-1.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

G-proteins in rat blood vessels--I. Identification.

1. The purpose of this investigation was to identify various types of conventional, low and high molecular weight G-proteins in purified membranes of rat aorta and mesenteric artery. 2. In both blood vessels, immunoblotting of G-proteins using AS/7 antibody specific for Gi-1/2, EC/2 antibody specific for Gi-3 and RM/1 antibody specific for Gs-type conventional G-proteins demonstrated the presence of M(r) 28-43 kDa, M(r) 41 and 48 kDa, and M(r) 36-46 kDa polypeptides, respectively. Immunoblotting using a common antibody (GA/1) for the Gs and Gi alpha-subunits also revealed the existence of multiple polypeptides (M(r) 24-52 kDa) in both aorta and mesenteric artery, some of which were identified by the specific antibodies. The intensity and number of bands detected by most of the antibodies were greater in mesenteric artery than in aorta. 3. Cholera toxin (CT) catalyzed ADP-ribosylation of two Gs alpha (M(r) 43, 46 kDa) in both types of blood vessels with similar intensities of bands. Pertussis toxin (PT), on the other hand, catalyzed ADP-ribosylation of one Gi alpha (M(r) 41 kDa) polypeptide, and the intensity of this band was greater in aorta than in mesenteric artery. The polypeptides ADP-ribosylated by the toxins corresponded with their identification by antibodies. 4. Nitrocellulose blot overlay with [35S]GTP gamma S identified at least seven low molecular weight G-proteins (M(r) 21-30 kDa) in both aorta and mesenteric artery, with greater intensity of bands in mesenteric artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗

PubMed1995

G-proteins in rat blood vessels--II. Assessment of functional involvement.

1. In this study, we compared G-protein-mediated contractile responses of rat aorta and mesenteric artery rings induced by the alpha-adrenoceptor agonist, norepinephrine (NE) and by the direct G-protein activator, sodium fluoride, using various probes. 2. Activator of the stimulatory G-protein (Gs), cholera toxin (CT), attenuated the sensitivity and maximum contractile response of both aorta and mesenteric artery to NE and sodium fluoride. The effect of the toxin on the NE-sensitivity was greater in mesenteric artery. 3. Pretreatment of tissues with the inhibitor of Gi-protein, pertussis toxin (PT), reduced the sensitivity as well as maximum contraction of both the aorta and mesenteric artery to sodium fluoride, and of the mesenteric artery to NE. PT attenuated only the sensitivity but not the maximum contraction of the aorta to NE. The inhibitory effect of PT on sensitivity to NE or sodium fluoride was greater in the aorta. 4. NE and sodium fluoride-induced contractions were reduced by the sulfhydryl G-protein inhibitor, N-ethylmaleimide (NEM) in both blood vessels. NEM produced greater inhibitory effect on the sensitivity of the aorta to both contractile agents. 5. These data demonstrate that CT, PT and NEM-sensitive G-proteins are involved in NE- and sodium fluoride-induced contractile responses of the rat aorta and mesenteric artery. The differential effects of the G-protein probes indicate that certain variations in G-protein-mediated contractile responses exist among the two blood vessels, suggesting that G-protein involvement in functional responses may vary with the type of blood vessel investigated.

Animals↗

PubMed1995

Role of tyrosine kinases in norepinephrine-induced contraction of vascular smooth muscle.

We used various probes to examine the involvement of tyrosine kinases in norepinephrine (NE)-induced contractile responses of the isolated rat aorta denuded of endothelium. The putative tyrosine kinase inhibitors, genistein and tyrphostin, significantly inhibited the contractile responses of the aorta to NE but not to potassium chloride (KCl). The protein tyrosine phosphatase inhibitor, sodium orthovanadate, also selectively potentiated the contractile response of the artery to NE. The inhibitory effect of genistein on NE-induced contraction was observed both in the absence and presence of extracellular calcium, which produced phasic and tonic contractile responses, respectively. The effect of genistein was more pronounced on the phasic contraction, suggesting that tyrosine kinases play a greater role in mediating the responses associated with the release of intracellular calcium. Genistein, however, had no effect on contraction elicited by the direct protein kinase C (PKC) activator phorbol 12, 13 dibutyrate (PDB), providing evidence for the lack of involvement of tyrosine kinases in PKC-mediated contractile responses which contribute to the NE-induced tonic contraction. In contrast, genistein attenuated the contraction of the vessel evoked by the direct G-protein activator sodium fluoride (NaF), suggesting the involvement of tyrosine kinases in responses associated with G-protein activation. The data indicate that genistein- and tyrphostin-sensitive tyrosine kinases participate in NE-induced contraction of rat aortic smooth muscle. Although this may involve one or more steps in the signal transduction pathway, the enzymes appear to have a greater role in mediating the responses linked to the release of intracellular calcium and have no roles in certain other processes, including those mediated by PKC activation.

Animals↗

PubMed1994

Cigarette smoking increases monocyte adherence to cultured endothelial cell monolayer.

Leukocyte adherence to endothelium represents one of the early responses to injury. Although the underlying mechanisms of atherosclerosis and intimal hyperplasia remain unclear, monocytes/macrophages are associated with this phenomenon. Since cigarette smoking has been identified as a major risk factor for cardiovascular disease, we examined the effects of chronic cigarette smoking on human leukocyte adherence (monocytes, lymphocytes, and neutrophils) to cultured human umbilical vein endothelial cells (HUVEC's) in vitro. Among the cells, the basal adherence of monocytes only was significantly elevated in the smoking group (p < 0.05) compared to the non-smokers. Furthermore, the increase is related to increasing pack per day consumption (p < 0.01). These novel data suggest an alteration of the circulating monocytes by cigarette smoking, resulting in increased adherence to endothelial cells.

Cell Adhesion↗

PubMed1994

Attenuation of human leukocyte adherence to endothelial cell monolayers by tyrosine kinase inhibitors.

Expression of cell adhesion molecules is controlled by various cytokines including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha), but the underlying mechanisms are not clear. In this study, we investigated the role of tyrosine phosphorylated proteins in the regulation of leukocyte adhesion to endothelial cells. We examined the effect of genistein and herbimycin-A, selective inhibitors of tyrosine kinase, on the adherence of human blood neutrophils, lymphocytes, and monocytes to monolayers of human umbilical vein endothelial cells. Both genistein and herbimycin A significantly inhibited IL-1 and TNF alpha-induced upregulation of neutrophils (p < 0.05) and monocyte (p < 0.01) adherence. IL-1 and TNF alpha-stimulated lymphocyte adherence was diminished in the presence of herbimycin A (p < 0.05), but genistein only inhibited TNF alpha-stimulated adherence. There was no significant effect of genistein on IL-1-induced lymphocyte adherence. These novel findings reveal for the first time that tyrosine phosphorylated proteins may regulate leukocyte adherence and cell adhesion molecule expression on the endothelium.

Analysis of Variance↗

PubMed1994

Neuropeptide Y levels during ischemia and reperfusion in the canine infrarenal aortic revascularization model.

Neuropeptide Y (NPY) is a potent vasoconstrictive polypeptide colocalized with norepinephrine in sympathetic neurons. The effects of ischemia and reperfusion on plasma NPY levels were studied and compared in the mongrel dog after infrarenal aortic cross-clamping. We found a two- to threefold increase in NPY levels during ischemia (initial 10.0 +/- 1.8 pmol/L vs. maximum 24.7 +/- 2.31 pmole/L, p < 0.001). The increase in NPY remained following reperfusion (initial 10.0 +/- 0.8 pmole/L vs. maximum 23.9 +/- 2.31 pmole/L, p < 0.001). These data reveal that NPY is released during ischemia and reperfusion and may be involved in mediating remote vascular events associated with infrarenal aortic cross-clamping.

Animals↗

PubMed1994

Technique for assessment of leukocyte adherence to human umbilical vein endothelial cell monolayers.

The interaction between endothelial cells and immune/inflammatory cells plays an important role in the pathogenesis of vascular diseases. Inflammatory cells also activate endothelial cells and release both proliferative and cytotoxic mediators. In order to examine the interaction between leukocytes and endothelial cells and the effect of various drugs, we established the methodology for isolating and culturing the endothelial cells from human umbilical vein. Endothelial cells were harvested by using 0.1% collagenase within 48 hr of collecting the cord. Cells were grown to confluency in 96-well plates in Medium 199 containing 20% fetal calf serum, endothelial cell growth supplement, heparin, and antibiotics. Using this method, we obtained a confluent layer of the cells in all the 96 wells within 48 hr. We then examined the effect of peptides, endothelin-1, substance P, and neurokinin-A on the adherence of human blood neutrophils (purity and viability > 98%) to endothelial monolayers. All the peptides enhanced (p < 0.05) the adherence of neutrophils to endothelial cells in a time-dependent manner. This method of endothelial cell culturing is reliable, reproducible, and effective in evaluating the role of various mediators and drugs on the adherence of various white blood cells to endothelium.

Cell Adhesion↗

PubMed1994

Guanine nucleotide binding regulatory proteins: their characteristics and identification.

Many biological signals are processed by the binding of chemicals to cell surface receptors. Signals are switched to intracellular language via guanine nucleotide binding regulatory proteins (G-proteins) which are present in all eukaryotic cells. Thus, G-proteins serve as interfaces between receptor-response coupling. Two forms of G-proteins have been reported: conventional G-proteins which are heterotrimeric and consist of alpha, beta, and gamma subunits, and monomeric small molecular weight G-proteins which are generally found as single polypeptides. Recently, high molecular weight G-proteins have also been described. The family of G-proteins contains multiple genes that encode the alpha, beta, or gamma subunits. G-proteins play a pivotal role in excitation-contraction coupling in smooth muscle function and control metabolic and secretory processes. In this review article, we have given a brief overview on the characteristics and methodology for the identification of G-proteins. The heterotrimeric G-proteins are generally identified by Western blotting and ADP-ribosylation with bacterial toxins. The monomeric and high molecular weight G-proteins have been identified by [35S]GTP delta S overlay technique and photoaffinity labeling, respectively. Recently, the use of molecular genetic probes has made it possible to investigate the expression of the message for various G-proteins.

Adenosine Diphosphate Ribose↗

PubMed1994

Endothelin-1 levels in ischaemia, reperfusion, and haemorrhagic shock in the canine infrarenal aortic revascularisation model.

Endothelin-1 (ET-1) is a potent vasoconstrictive polypeptide produced from vascular endothelial cells. The effects of ischaemia, reperfusion, and exsanguination on plasma ET-1 levels were studied and compared in the mongrel dog after infrarenal aortic cross clamping. Ischaemia produced a trend toward increased ET-1 serum levels (p < 0.07 with Bonferroni correction) that did not reach significance. Plasma ET-1 levels were significantly increased during reperfusion and even further elevations were found following exsanguination. We found a 2-3 fold increase in ET-1 levels following reperfusion (Initial 3.19 +/- 0.27 pg/ml vs. Reperfusion maximum 6.32 +/- 0.72 pg/ml, Bonferroni p < 0.01). Haemorrhagic shock was associated with a 3-4 fold increase in ET-1 levels (Initial 3.19 +/- 0.27 pg/ml vs. Exsanguination maximum 8.37 +/- 0.97 pg/ml Bonferroni p < 0.001). These data reveal that ET-1 is released during reperfusion and exsanguination and may mediate remote vascular events associated with infrarenal aortic cross clamping and acute blood loss.

Animals↗

PubMed1993

Posttraumatic innominate artery aneurysm with occlusion of the common carotid artery at its origin by an intimal flap.

Blunt trauma involving the innominate and carotid arteries is a rare occurrence that can be lethal or have serious neurologic sequelae. To our knowledge this is the first reported case in the international literature describing the association of posttraumatic innominate artery aneurysm with total occlusion and thrombosis of the common carotid artery at its origin by an intimal flap. The diagnostic problems created by this unusual injury are discussed. In this case the patency of the distal portion of the common and internal carotid arteries was demonstrated by magnetic resonance angiography (MRA), whereas color duplex and digital arteriographic studies were unsuccessful. This demonstration was crucial to patient management. Since no studies are available comparing color duplex imaging, conventional arteriography, and MRA in the evaluation of blunt carotid trauma, this case study is presented to demonstrate the utility of MRA in emergency situations. In addition, we analyze the possible pathogenesis and discuss the surgical treatment.

Adolescent↗

PubMed1993

Functional and morphological assessment of rat aorta stored in University of Wisconsin and Eurocollins solutions.

University of Wisconsin (UW) and Eurocollins (EC) solutions are widely used for preservation of organs before transplantation. However, effect of storage solutions on vascular interface for transplant success is not known. In this study, we have used rat aorta as a model and assessed the effects of cold storage in UW and EC solutions on smooth muscle and endothelial function and the morphology. Smooth muscle and endothelial functions of the rat aorta were assessed using in vitro isometric tension measurement. Morphologic studies were done with scanning and transmission electron microscopy. No significant difference in contractile response to either norepinephrine (NE) or potassium chloride was observed between control aorta and aorta stored in UW solution for 1 hr or 24 hr. In contrast, sensitivity, but not the reactivity to NE and KCl, was increased in aorta stored in EC solution for 1 hr. If the tissues were stored in EC solution for 24 hr, both sensitivity and reactivity to NE and KCl were significantly reduced. Relaxatory response to acetylcholine, in endothelium-intact vessels were reduced in aortas stored in EC solution, but not in UW solution. The magnitude of relaxations observed in tissues stored in the EC solution for 24 hr was less than in tissues stored for 1 hr. Sodium nitroprusside elicited similar relaxatory response in endothelium-denuded control tissue and in tissues stored in UW and EC solution. Electron microscopy data revealed marked swelling of the cell, loss of mitochondria and other intracellular organelles, and striking calcium deposits after preservation of the vessels in EC for 1 or 24 hr. In aorta stored in UW solution for 24 hr, endothelial and smooth muscle cells were intact, with moderate-size vacuoles in the cytoplasm. These results suggest that the UW solution is more suitable than EC solution for short-term preoperative storage of blood vessels.

Acetylcholine↗

PubMed1992

Neuropeptides modulate human eosinophil chemotaxis.

To investigate the role of neuropeptides in allergic inflammation, we examined the effect of peptides on eosinophil chemotaxis. Eosinophils were purified from the blood of allergic and normal subjects using a discontinuous Percoll density gradients. Chemotaxis was induced by platelet-activating factor (PAF) and leukotriene B4, and was assayed by a modified Boyden's chamber technique. Four neuropeptides were examined in this study: substance P (SP), neurokinin A, calcitonin gene-related peptide (CGRP), and cholecystokinin octapeptide. Peptides alone (10 nM to 10 microM) were not chemotactic for eosinophils. However, when eosinophils were pre-treated with peptides (100 nM) at 37 degrees C for 30 min, chemotactic response to PAF (10 nM) was significantly enhanced (p < 0.01) in allergic subjects; % control by SP, neurokinin A, CGRP and cholecystokinin octapeptide was 269 +/- 42, 243 +/- 32, 227 +/- 21, and 251 +/- 42, respectively (n = 8). Similar results were obtained in leukotriene B4-induced eosinophil chemotaxis. In contrast, no enhancement was observed in normal subjects. Potentiating effect of SP and CGRP on PAF-induced eosinophil chemotaxis in allergic subjects was significantly attenuated by SP antagonist [D-Pro2,D-Trp7,9]-SP and human CGRP (8-37) receptor antagonist, respectively. Neutral endopeptidase inhibitors (phosphoramidon, leupeptin, and bestatin) failed to significantly augment the PAF-induced eosinophil chemotaxis when the cells were pretreated with various peptides and neutral endopeptidase inhibitors. The C-terminal fragment of SP (SP6-11) had an effect similar to that of the intact SP molecule, whereas no potentiating effect by the N-terminal of SP (SP1-9) was observed. These results suggest that neuropeptides may play a significant role in eosinophil infiltration by priming cells in allergic inflammation.

Chemotaxis, Leukocyte↗

PubMed1992

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes.

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa Gs alpha, and 39 and 41 KDa Gi alpha subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [alpha-32P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160, 100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21-28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.

Amylases↗