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Biomedical subjects

M A Napier

Publications and source records attributed to M A Napier.

At least 19 recordsLinked to original sources
PubMed1991

Cloning and expression of a divergent integrin subunit beta 8.

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.

Amino Acid Sequence↗
PubMed1991

Kistrin, a polypeptide platelet GPIIb/IIIa receptor antagonist, enhances and sustains coronary arterial thrombolysis with recombinant tissue-type plasminogen activator in a canine preparation.

BACKGROUND: Kistrin is a 68-amino acid polypeptide from the venom of the Malayan pit viper Agkistrodon rhodostoma, which inhibits the platelet GPIIb/IIIa receptor. Its effect on thrombolysis, reocclusion, and bleeding associated with administration of recombinant tissue-type plasminogen activator (rt-PA) was studied in a canine model of coronary artery thrombosis. METHODS AND RESULTS: Coronary patency was monitored for 2 hours by ultrasonic flow probe and repeated coronary angiography. The rt-PA was given as 0.45-mg/kg bolus injections at 15-minute intervals until recanalization or to a maximum of four boluses. Four groups of four or five dogs were studied: a control group that received intravenous heparin (4,000-unit bolus and 1,000 units each hour) and three groups that received heparin and 0.48, 0.24, or 0.12 mg/kg kistrin, administered as a 10% bolus injection and an infusion during a 60-minute period. In the control group, reflow occurred in four of five dogs within 37 +/- 47 minutes but was followed by cyclic reflow and reocclusion. Kistrin at a dose of 0.48 and 0.24 mg/kg reduced the time to reflow to 6 +/- 5 and 10 +/- 3 minutes, respectively, and abolished reocclusion. With 0.12 mg/kg kistrin, reflow occurred in all four animals, within 27 +/- 23 minutes, and reocclusion occurred in two animals. Kistrin induced a dose-related prolongation of the template bleeding time: with 0.48 mg/kg kistrin, the bleeding time was prolonged from 3.8 +/- 1.3 minutes before infusion to 29 +/- 2 minutes during infusion, but it was shortened to 8.3 +/- 2.6 minutes at 90 minutes after the end of infusion. Kistrin also caused a dose-related inhibition of platelet aggregation with ADP and collagen: with 0.48 mg/kg kistrin, platelet aggregation was abolished during the infusion but had partially recovered toward the end of the observation period. Pathological examination of recanalized coronary arterial segments of dogs given 0.48 or 0.24 mg/kg kistrin revealed widely patent arteries with some platelets layered on the damaged intimal surface. CONCLUSIONS: Kistrin increases the rate and extent of thrombolysis with a reduced dose of rt-PA, and it prevents reocclusion. At an effective dose, it is associated with a transient prolongation of the bleeding time and inhibition of platelet aggregation. Kistrin may offer promise as adjunctive treatment to thrombolytic agents in patients with acute myocardial infarction.

Animals↗
PubMed1991

Characterization of an anti-p185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell growth.

The HER2 protooncogene encodes a growth factor receptor-like transmembrane protein tyrosine kinase (p185HER2) whose ligand remains to be fully characterized. The overexpression of p185HER2 is implicated in aggressive forms of breast and ovarian cancers. The role of p185HER2 in aggressive malignancy, as well as its cell surface localization, makes it an attractive target for therapeutic monoclonal antibodies. In this report we have studied the modulation of p185HER2 function with 2 monoclonal antibodies, termed 4D5 and 6E9, which bind the extracellular domain of p185HER2. 4D5 inhibited proliferation of p185HER2 overexpressing SK-BR-3 human breast carcinoma cells (ED50 of approximately 1 nM) but did not inhibit proliferation of cultured human breast carcinoma MCF7 cells, low expressors of p185HER2. Monoclonal antibody 6E9 does not inhibit the growth of either cell line. Antibody binding studies revealed 2 populations of p185HER2 molecules on SK-BR-3 cells: one of high abundance (approximately 2 x 10(6) sites/cell) recognized by 4D5 (Kd approximately 6 nM) and the other of low abundance (2 x 10(4) sites/cell) recognized by 6E9 (Kd approximately 0.1 nM). 4D5, in an agonistic manner, downregulated SK-BR-3 cell surface p185HER2, was internalized, and stimulated p185HER2 phosphorylation in intact cells. Phosphoamino acid analysis of p185HER2 derived from SK-BR-3 cells incubated with the 4D5 monoclonal antibody demonstrated increased tyrosine, serine and threonine phosphorylation. 4D5, on short term (5 min) exposure to SK-BR-3 cells, stimulated inositol lipid hydrolysis as evidenced by increased intracellular levels of inositol polyphosphates (InsP) and sn-1,2-diacylglycerol (sn-1,2-DAG). On longer (24 h) exposure to the cells, the antibody appeared to downregulate this signalling pathway since the intracellular levels of InsP and sn-1,2-DAG decreased by 30 to 40%. 6E9 did not inhibit SK-BR-3 cell proliferation, downregulate surface p185HER2, stimulate receptor phosphorylation, or stimulate the second messenger pathway. Despite these agonistic properties, 4D5 was an inhibitor of SK-BR-3 cell proliferation at all concentrations tested (0.7 to 70 pM). The data suggest that 4D5 is a partial or weak agonist and thus may inhibit cell proliferation by mimicking ligand-like receptor downregulation.

Antibodies, Monoclonal↗
PubMed1990

Characterization of murine monoclonal antibodies reactive to either the human epidermal growth factor receptor or HER2/neu gene product.

High levels of expression of either the epidermal growth factor receptor or the receptor-like HER2/neu gene product p185HER2 have been observed in a variety of human malignancies. Because of the association of this high level expression with certain human tumors, we have generated a panel of monoclonal antibodies specific for either the epidermal growth factor receptor or p185HER2 to study their structure, function, and antigenic domains in the normal and neoplastic states. We used the epidermoid carcinoma line A431 to generate five monoclonal antibodies which immunoprecipitate the epidermal growth factor receptor. These monoclonal antibodies bind to the extracellular domain of the epidermal growth factor receptor and demonstrate variable effects on epidermal growth factor binding. We used a stably transfected NIH 3T3 cell line expressing the HER2/neu gene to produce and characterize 10 monoclonal antibodies which immunoprecipitate p185HER2. These monoclonal antibodies bind to the extracellular domain of p185HER2 and do not cross-react with the epidermal growth factor receptor. The characteristics and potential applications of these monoclonal antibodies will be discussed.

Antibodies, Monoclonal↗
PubMed1990

Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors.

The purification, complete amino acid sequence, and biological activity are described for several homologous snake venom proteins that are platelet glycoprotein (GP) IIb-IIIa antagonists and potent inhibitors of platelet aggregation. The primary structures of kistrin (from Agkistrodon rhodostoma), bitan (from Bitis arietans), three isoforms of trigramin (from Trimeresusus gramineus), and an isoform of echistatin (from Echis carinatus) were determined by automated sequence analysis and fast atom bombardment mass spectrometry analysis. Each of the protein in this family, which range from 47 to 83 residues, contains an Arg-Gly-Asp amino acid sequence found in protein ligands that binds to GPIIb-IIIa, a high (17 +/- 1%) cysteine content conserved in the primary sequence, and a homologous N-terminal region absent only in the echistatin isoforms. Each protein directly inhibits the interaction of purified platelet GPIIb-IIIa to immobilized fibrinogen about 100 times more effectively than does the pentapeptide Gly-Arg-Gly-Asp-Ser; IC50 values range from 1.1 to 3.0 nM. The IC50 value for the inhibition of platelet aggregation, using human platelet-rich plasma stimulated with ADP, ranges from 110 to 550 nM for the various proteins, about 1000-fold more potent than Gly-Arg-Gly-Asp-Ser. Kistrin binds reversibly to both resting and ADP-activated human platelets with high affinity (Kd = 10.8 nM and 1.7 nM, respectively) and to purified GPIIb-IIIa with a lower affinity (Kd = approximately 100 nM). Finally, kistrin injected at 1.0 mg/kg into rabbits reversibly inhibits platelet aggregation ex vivo over 30 min without induction of thrombocytopenia. We propose that these proteins are members of a general class of proteins found in the venom of pit vipers that inhibit platelet aggregation by antagonism of the GPIIb-IIIa-fibrinogen interaction and as such serve as potential antithrombotic agents.

Amino Acid Sequence↗
PubMed1990

Growth hormone stimulates the formation of sn-1,2-diacylglycerol in rat hepatocytes.

The transmembrane signaling events of GH were investigated in the liver, a major target organ of GH action. Recombinant human GH when added to freshly isolated rat hepatocytes rapidly stimulated the production of sn-1,2-diacylglycerol (DAG). The generation of DAG was biphasic with the first transient peak observed at 2 min and the second peak at 15 min (1.2-fold and 1.4-fold over control, respectively). Levels of DAG continued to be elevated above those in control cells at 30 min. The response was dose-dependent with an EC50 of 0.15 nM. Both bovine GH and rat GH, which bind to the rat GH receptor but not to the PRL receptor, also stimulated DAG production. Similarly, human PRL, which binds to the PRL but not GH receptor, stimulated DAG formation to a comparable extent. These results suggest that production of DAG may be an early signaling event mediated by hormone stimulation of both the GH and PRL receptors.

Animals↗
PubMed1989

Expression of recombinant platelet glycoprotein IIbIIIa results in a functional fibrinogen-binding complex.

The ability of cDNAs encoding the human platelet glycoprotein IIbIIIa to be expressed and assembled into a functional integrin receptor was assessed by transient transfection into a human cell line. Transfection of full length cDNAs resulted in synthesis of high levels of integrin subunits which appear to be stable within the cell for several days. Coexpression of both subunits resulted in a proteolytically processed form of GPIIb that associated with GPIIIa as a heterodimeric complex as the cell surface. Transport to the cell surface required association of these subunits with each other or with endogenous integrin subunits. When expressed alone, the GPIIb subunit remained intracellular, while the GPIIIa subunit was found to complex with endogenous proteins and was mobilized to the cell surface. The GPIIbIIIa receptor complex facilitated attachment of cells to known ligands for GPIIbIIIa: fibrinogen, vitronectin, and von Willebrand factor. This adhesion was sensitive to inhibition by the peptide GRGDV and the monoclonal antibody AP2, known inhibitors of platelet aggregation

Base Sequence↗
PubMed1987

Epidermal growth factor receptor tyrosine kinase phosphorylation of glucose-6-phosphate dehydrogenase in vitro.

The specific tyrosine phosphorylation of glucose-6-phosphate dehydrogenase (G6PDH) by the epidermal growth factor (EGF) receptor in vitro is demonstrated. The Km values of the substrate G6PDH and of ATP for the receptor tyrosine kinase were ca. 1 and 10 microM, respectively. The rate of phosphorylation was EGF dependent, with a four-fold increase in Vmax in the presence of EGF. The phosphorylation was stimulated maximally by 0.2 microM or greater EGF, with an ED50 of ca. 20 nM which is consistent with the affinity of the solubilized receptor for EGF. Using conditions of 5 microM G6PDH, 100 microM ATP, 5 mM Mg2+, and 1 mM Mn2+, up to 0.3 mol phosphate was incorporated into 1 mol of the 55-kDa subunit of Baker's yeast G6PDH. Tryptic peptide mapping revealed several unique phosphopeptides for both Baker's yeast and bovine adrenal G6PDH. The patterns of phosphopeptides for a given enzyme were identical for basal and EGF-stimulated phosphorylation.

Animals↗
PubMed1986

Binding and internalization of atrial natriuretic factor by high-affinity receptors in A10 smooth muscle cells.

A10 smooth muscle cells, derived from embryonic rat thoracic aorta, responded to the atrial natriuretic factor (ANF) with increased levels of cyclic GMP. These cells possess high-affinity (apparent Kd = 50 pM) plasma membrane receptors for ANF. Internalization of ANF at 37 degrees C was indicated by the following: approximately 25% of the 125I-ANF associated with the cells at elevated temperatures could not be dissociated from the surface of the cells, but could be released by permeabilization with saponin, and the amount of nondissociable ANF increased in the presence of chloroquine. In whole cells and in membranes, a single polypeptide of 60,000 Da was specifically labeled by a photoaffinity analog of 125I-ANF, as well as by crosslinking, and an IC50 of 80 pM for inhibition of the labeling by ANF was observed. The ANF receptor in A10 cells was distinguished from that in rabbit aorta by its high affinity for shorter and linear analogs of ANF, as well as by a different photolabeling pattern.

Animals↗
PubMed1986

Effects of atrial natriuretic factor on renal function and cyclic GMP production.

Anesthetized beagle dogs received increasing doses of continuous infusions of a 26-amino-acid synthetic atrial natriuretic factor (ANF). Urinary sodium excretion rose in a dose-dependent manner to a maximum level similar to that seen after hydrochlorothiazide administration. Mean arterial blood pressure decreased, but only modestly, and not in a dose-dependent fashion. Dogs chronically retaining NaCl secondary to constriction of the thoracic inferior vena cava showed only modestly enhanced natriuresis when infused with similar levels of ANF. When ANF was infused directly into the renal artery of anesthetized beagles, a dose-dependent natriuresis and calciuresis were observed with maximal fractional sodium excretion averaging approximately 8%. Although glomerular filtration tended to increase, the average dose-related changes were not significant. Cyclic GMP excretion was increased during intra-renal-arterial infusion of ANF. Excretion of cyclic GMP by both the infused and noninfused kidneys was equal, which suggests that urinary cyclic GMP was not nephrogenous but derived from the elevated circulating levels. These and other data from rats dissociate changes in urinary cyclic GMP excretion and sodium excretion.

Animals↗
PubMed1986

Molecular characteristics of receptors for atrial natriuretic factor.

Specific, high-affinity receptors for atrial natriuretic factor (ANF) have been identified on membranes from a variety of tissues and cultured cells. By affinity labeling procedures, radioactivity from 125I-labeled ANF was specifically incorporated into three different polypeptides of ca. 120,000, 70,000, and 60,000 daltons, which may represent the binding subunits of ANF receptors. These polypeptides were present in varying amounts in different target tissues. In rat adrenal membranes, the 120,000- and 70,000-dalton peptides were specifically labeled whereas in A10 rat smooth muscle cells, only the 60,000-dalton peptide was labeled. Membranes from rat kidney and rabbit aorta contain all three peptides. Gel filtration chromatography of solubilized receptors suggested that intact ANF receptors are large molecular complexes with apparent molecular masses in the range of 250,000-350,000 daltons. The differential labeling pattern observed with the various tissues suggested that there might be at least two different receptors composed of unique ANF-binding polypeptides.

Adrenal Glands↗
PubMed1985

Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking.

Binding sites in rabbit aorta membranes for atrial natriuretic factor (ANF) have been specifically and covalently labeled by two methods. In the first, the photoreactive analog of ANF, 125I-azidobenzoyl-ANF, was synthesized and used to photoaffinity label ANF receptors. In the second, 125I-ANF was covalently attached to its binding site by treatment of the 125I-ANF-receptor complex with bifunctional cross-linking agents. Analysis of the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that by both methods the same three protein bands were labeled. These bands had apparent molecular masses of 60,000, 70,000, and 120,000 daltons. With the photoaffinity label, half-maximal inhibition of labeling of each of these bands was achieved when approximately 200 pM of unlabeled ANF was included in the binding assay. These results suggest that these three different polypeptides are specific components of ANF receptors in rabbit aorta membranes.

Affinity Labels↗
PubMed1985

Renal and systemic effects of synthetic atrial natriuretic factor.

A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat atrial natriuretic factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted directly on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not significantly affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat atria and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance fell progressively as doses of ANF were increased. Glomerular filtration rate was not significantly elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean arterial pressure was progressively reduced, there was no significant change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concentration was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in circulating concentrations of arterial cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP filtration, by enhanced production in tubular cells, or by renal tubular secretion. Especially in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels.

Animals↗
PubMed1985

Pharmacology and receptor binding of atrial natriuretic factor in vascular smooth muscle.

Characterization of the vascular pharmacology and receptor binding of atrial natriuretic factor (ANF) has been achieved utilizing a synthetic peptide which contains the sequence and biological activity of ANF. The synthetic ANF (sANF) relaxes aortic segments contracted by agonists or by low (15 to 20 mM) but not high (greater than or equal to 40 mM) concentrations of K+. The relaxation to sANF is well maintained, reversible, independent of the vascular endothelium and correlated with increases in cyclic GMP (with no change in cyclic AMP). Plasma membranes prepared from rabbit aorta and kidney possess high affinity (Kd = 100 pM) specific binding sites for sANF. An excellent correlation exists between the receptor binding and pharmacology for several synthetic analogs of ANF. The presence of these receptors appears consistent with the activation of particulate (but not soluble) guanylate cyclase by sANF. sANF does exhibit a profound regional vasodilator selectivity which can be explained, in part, by changes in receptor density.

Amino Acid Sequence↗
PubMed1978

Effect of calcium on structure and function of a hyaluronic acid matrix: carbon-13 nuclear magnetic resonance analysis and the diffusional behavior of small solutes.

Natural-abundance (13)C NMR at 25.16 MHz has been used to study a 2.5% matrix of hyaluronic acid at various degrees of polymerization and at various ionic strengths. Peak assignment is facilitated by comparing proton-decoupled and off-resonance-decoupled spectra of a hyaluronidase-depolymerized matrix with spectra from relevant monosaccharides. In contrast to the spectrum following depolymerization, the spectrum for intact matrix has considerable broadening, particularly for peaks assigned to the N-acetylglucosamine moiety. This is most dramatic for the hydroxymethylene carbon. With the addition of Ca(2+) above 5 mM these broadened peaks narrow and approach the sharpness observed for the hyaluronidase digest. There is no shift in resonance peak positions. These changes are quantitatively less impressive if Na(+) is substituted for Ca(2+). The data suggest the existence of a considerable degree of order in regions of the matrix at physiological concentrations of Ca(2+). Within such a matrix the translational movement of lysine and glucose is enhanced relative to that in a matrix of agarose. Further addition of Ca(2+) abrogates not only matrix order, but the enhanced diffusivity as well.

Calcium↗
PubMedDate not supplied

Polyclonal hyperviscosity syndrome.

The clinical course of two patients who presented with polyclonal hyperviscosity syndrome is described. Polymerizing IgG rheumatoid factors were isolated from the serum of both patients. The presence of these polymers with their extraordinary rheologic properties was the principal determinant of the abnormal rheological properties of the plasma. Neither patient had a discrete rheumatic disease. In both, a lymphoproliferative syndrome was present with features suggestive of the "pseudolymphoma" of Sjögren's syndrome.

Blood Viscosity↗