Liposome-mediated transfection of intact viral particles reveals that plasma membrane penetration determines permissivity of tissue culture cells to rotavirus. Rotaviruses are an important cause of gastroenteritis in human infants. In vivo, rotavirus displays striking cell tropism with viral replication generally restricted to the villus tip enterocytes of the small intestine. We studied a panel of cell lines that vary significantly in their permissivity to rotavirus infection. L cells and HEp2 cells were relatively resistant to rotavirus infection compared with permissive Ma104 cells and HT29 cells. RNA transcription among the cell lines was proportional to antigen synthesis making a translational or posttranslational block an unlikely source of observed differences in susceptibility. All of the cell lines bound and internalized radiolabeled virus equally well, as measured by escape from surface protease treatment. Analysis of the escape of cell bound virus from neutralizing monoclonal antibody revealed that rotavirus did not immediately enter an eclipse phase in nonpermissive cells, but was internalized in an infectious form for several hours, possibly sequestered within endocytic vacuoles. L cells and HEp2 cells were as permissive as Ma104 and HT29 cells when rotavirus infection was mediated by transfection of single- or double-shelled rotavirus particles with cationic liposomes (Lipofectin). Rotavirus cell tropism in tissue culture cells is determined by the ability of infecting virions to traverse the plasma membrane of the cells into the cytoplasmic compartment.


Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide. Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.


Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells. Renal proximal tubule sodium reabsorption is enhanced by apical or basolateral angiotensin II (AII). Although AII activates phospholipase C (PLC) in other tissues, AII coupling to PLC on either apical or basolateral surfaces of proximal tubule cells is unclear. To determine if AII causes PLC activation, and the differences between apical and basolateral AII receptor function, receptors were unilaterally activated in rat proximal tubule cells cultured on permeable, collagen-coated supports. Apical AII incubation resulted in concentration- and time-dependent inositol trisphosphate (IP3) formation. Basolateral AII caused greater IP3 responses. Apical AII-induced IP3 generation was inhibited by DuP 753, suggesting that the type 1 AII receptor subtype mediated proximal tubule PLC activation. Apical AII signaling did not result from paracellular ligand leak to basolateral receptors since AII-induced PLC activation occurred when basolateral AII receptors were occupied by Sar-Leu AII or DuP 753. Inhibition of endocytosis with phenylarsine oxide prevented apical (but not basolateral) AII-induced IP3 formation. Cytoskeletal disruption with colchicine or cytochalasin D also prevented apical AII-induced IP3 generation. These results demonstrate that in cultured rat proximal tubule cells, AII is coupled to PLC via type 1 AII receptors and cytoskeleton-dependent endocytosis is required for apical (but not basolateral) AII receptor-mediated PLC activation.


Immunopotentiating reconstituted influenza virus virosome vaccine delivery system for immunization against hepatitis A. Hepatitis A virus (HAV) was purified from MRC-5 human diploid cell cultures, inactivated with formalin, and evaluated for safety and immunogenicity in humans. Three vaccine formulations were produced: (a) a fluid preparation containing inactivated HAV, (b) inactivated HAV adsorbed to Al(OH)3, and (c) inactivated HAV coupled to novel immunopotentiating reconstituted influenza virosomes (IRIV). IRIV were prepared by combining phosphatidylcholine, phosphatidylethanolamine, phospholipids originating from the influenza virus envelope, influenza virus hemagglutinin, and neuraminidase. The HAV-IRIV appeared as unilamellar vesicles with a diameter of approximately 150 nm when viewed by transmission electron microscopy. Upon intramuscular injection, the alum-adsorbed vaccine was associated with significantly (P < 0.01) more local adverse reactions than either the fluid or IRIV formulations. 14 d after a single dose of vaccine, all the recipients of the IRIV formulation seroconverted (> or = 20 mIU/ml) versus 30 and 44% for those who received the fluid and alum-adsorbed vaccines, respectively (P < 0.001). The geometric mean anti-HAV antibody titer achieved after immunization with the IRIV-HAV vaccine was also significantly higher (P < 0.005) compared with the other two vaccines.


Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation. Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.


Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. Hydrogen peroxide (H2O2)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H2O2 and other oxidant species generated from H2O2. We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis. Within minutes, H2O2 caused DNA strand breaks in a dose-dependent manner, followed by cell death. DNA fragmentation was demonstrated both by the release of [3H]thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage. Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H2O2-induced DNA strand breaks, fragmentation, and cell death. Inhibitors of protein or mRNA synthesis had only minor protection against H2O2-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes. Micrococcal endonuclease induced similar DNA strand breaks in LLC-PK1 cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage. The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium. We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis.


Entorhinal cortex of the human, monkey, and rat: metabolic map as revealed by cytochrome oxidase. The entorhinal cortex (EC) is a medial temporal lobe area involved in memory consolidation. Results from previous studies suggest that the upper layers of the EC may be organized into anatomical-neurochemical modules associated with pathways through the neuron clusters in layers II and III. To study metabolic patterns in the EC and to look for correlates of the proposed modules, we examined the distribution of cytochrome oxidase (CO) in the human, monkey, and rat EC. CO is a mitochondrial enzyme that has been used to study modules in other cortical areas. In all three species, the neuron clusters in layers II-III were darkly CO-reactive, whereas most of the neuropil between clusters was lightly or moderately CO-reactive. However, some neuropil regions directly adjacent to the neuron clusters were also darkly CO-reactive, especially in the human; these neuropil areas included portions of layers I and II. In tangential sections through layers I-II, the areas of dark staining formed a consistent pattern, comprised of partially interconnected islands and stripes associated with the neuron clusters. In the EC from one human hemisphere, approximately 200-250 CO-reactive layer II islands were present. EC layers other than I-III also showed characteristic CO staining intensities, but no evidence of modularity. Our results indicate that CO staining labels distinct compartments related to the neuron clusters in the upper EC layers. We propose that these compartments may represent modules for cortical processing, analogous to the CO-labeled modules in some other areas of cortex.


Effects of fowl adenovirus infection on the immune system of chickens. A virulent strain of serotype 8 fowl adenovirus (FAV) was isolated from an outbreak of inclusion body hepatitis (IBH) in broiler flocks. Post-mortem changes included characteristic liver lesions with intranuclear inclusion bodies in the hepatocytes and severe lymphocytic depletion in the bursa, thymus and spleen. The packed cell volume was reduced by 50 per cent or more and varying amounts of cell depletion were observed in the bone marrow. Typical IBH was reproduced in specific pathogen-free chickens inoculated orally with the FAV isolated from the natural infection. There was severe depletion of lymphocytes in the bursa, thymus and spleen of the experimentally infected birds and FAV antigens were detected by ELISA and immunocytochemical staining in various lymphoid tissues. Humoral antibody responses against sheep red blood cells, detected by the haemagglutination test, were decreased in the chickens infected with FAV. These findings suggest that the damage caused by replication of this virulent strain of FAV in lymphoid tissues compromises the immunological capabilities of infected chickens.


Detection of rabbit haemorrhagic disease virus particles in the rabbit liver tissues. Liver tissues from rabbits experimentally infected with rabbit haemorrhagic disease virus (RHDV) were studied electron microscopically. The earliest change in hepatocytes of the rabbits infected with RHDV was hydropic degeneration. Rough endoplasmic reticulum was dilated with a mild increase in polysomes and cytoplasmic cisternae in degenerated hepatocytes. Characteristic cytopathological changes of necrotic hepatocytes included shrinkage of the cell body, formation of cytoplasmic vesicles, vacuoles or cisternae and karyolysis. A large number of viral particles resembling a calicivirus in size and morphology was demonstrated in the cytoplasm of many necrotic hepatocytes. The particles had accumulated mainly in the membrane-bound cisternae or scattered around the membrane-bound vacuoles of the necrotic hepatocytes. Western blot analysis demonstrated that RHDV antigen was present in the infected hepatocytes. RHDV particles were also detected by immunoelectron microscopy. Replicating patterns of RHDV particles and subsequent cytopathology resembled those in other calicivirus infections.


Human papillomavirus type 6 infection involving cutaneous nongenital sites. Human papillomavirus (HPV) type 6 is classically considered a mucosatropic virus. Interestingly, clinical manifestations of HPV 6 infection that involve nonmucosal or nongenital sites have rarely been described. The reasons for this site specific infectivity of HPV 6 are unknown. We describe a patient who had condylomata acuminata-like lesions that involved cutaneous nongenital sites; HPV 6 DNA was detected in skin biopsy specimens with use of the polymerase chain reaction, followed by hybridization with use of type-specific DNA probes.


[Cytomegalovirus and pregnancy. Fetal and neonatal risk]. A review of the literature shows that 36% of pregnant women are not immunized against cytomegalovirus; 2.11% of these patients will have a primary infection during their pregnancy. The fetal transmission rate is 30%. Viruria after delivery is the unique proven sign of infection; but is only present in 4 to 5 infants per 1,000 births. Of those, 17% will be symptomatic at delivery and 10% will develop sequels as late as seven years later. CMV can be reactivated during pregnancy. These cases are less dangerous but the fetal risk is always present.


Increased rate of tuberculin skin test conversion among workers at a university hospital. To summarize the results of an investigation of increased rates of tuberculin skin test conversion in employees at a university hospital. The results of annual tuberculin skin tests performed on all 1,845 hospital employees from 1986 to 1991 were reviewed. A 450-bed acute tertiary care university hospital. The rate of tuberculin skin test conversion was 0.35% (standard deviation +/- 0.15) from 1986 to 1989 and increased to 1.7% during 1991. Investigation revealed deviations from the Centers for Disease Control (CDC) guidelines for tuberculosis control, which included the failure to consider tuberculosis as a probable cause of community-acquired pneumonia and the failure to initiate isolation precautions when tuberculosis was suspected. The epidemic appeared to be secondary to delays in diagnosis and isolation of patients with pulmonary tuberculosis. Future control measures should include isolation of all hospital patients admitted with pneumonia until tuberculosis has been excluded.


Consensus paper on the surveillance of surgical wound infections. The Society for Hospital Epidemiology of America; The Association for Practitioners in Infection Control; The Centers for Disease Control; The Surgical Infection Society. A Surgical Wound Infection (SWI) Task Force was convened by The Society for Hospital Epidemiology of America (SHEA) to evaluate how SWI surveillance should be done and to identify where more information is needed. The Task Force reached consensus in the following areas. The Centers for Disease Control (CDC) definitions of SWI should be used for routine surveillance because of their current widespread acceptance and reproducibility. The CDC definitions have been clarified in an accompanying article ("Report From the CDC"). Direct observation of wounds and traditional infection control surveillance techniques are acceptable methods of case finding for hospitalized patients. The optimal method for case finding postdischarge or after outpatient surgery is unknown at this time. SWI rates should be stratified by surgical wound class plus a measure of patient susceptibility to infection, such as the American Society of Anesthesiology (ASA) class, and duration of surgery. Surgeon-specific SWI rates should be calculated and reported to individual surgeons.


Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneous hypertensive cardiomyopathy in rats. The question of this study was whether alterations in the inhibitory guanine-nucleotide binding protein alpha-subunits (G(i)alpha) contribute to alterations in adenylate cyclase regulation in hypertensive cardiomyopathy of spontaneously hypertensive rats (SHR). G(i)alpha was measured by pertussis toxin-catalysed 32P-adenosine 5'-pyrophosphate (ADP)-ribosylation and radioimmunochemically by competition of rat myocardial membrane extracts to DS 4 antiserum binding to the 125I-radiolabelled C terminus of retinal transducin alpha (125I-KENLKDCGLF). Cardiac beta-adrenoceptors, m-cholinoceptors as well as isoprenaline-, guanine-nucleotide [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activity and inotropic responses to isoprenaline and carbachol were studied in SHR and age-matched Wistar-Kyoto (WKY, control) rats. In native membranes of SHR there was an increase in pertussis toxin substrates, but a larger increase in the presence of non-ionic detergent Lubrol PX. The radioimmunological quantification of G(i)alpha revealed an increase in membrane extracts of SHR. In addition, myocardial beta-adrenoceptors and myocardial m-cholinoceptors were reduced in SHR compared with in WKY rats. Basal adenylate cyclase, isoprenaline-, Gpp(NH)p- and forskolin-stimulated adenylate cyclase activities were also reduced. However, in the presence of 5 mmol/l MnCl2 no differences in adenylate cyclase activities between SHR and WKY rats were detected under either condition. The present study shows that the amount of G(i)alpha-proteins and not only pertussis toxin substrates are increased in membranes of hypertrophic hearts from SHR without heart failure. The results obtained with pertussis-labelling depended strongly on the substrate quality of G(i)alpha. Increased G(i)alpha expression and reduced beta-adrenoceptor number might have functional relevance in the regulation of adenylate cyclase activity and force of contraction in SHR. An increase in G(i)alpha expression might play a pathophysiological role, not only in terminal heart failure, but also in hypertrophic cardiomyopathy.


Role of the renal medulla in adrenocorticotrophin-induced hypertension in rats. The mechanism of hypertension induced by adrenocorticotrophin (ACTH) remains unclear. The antihypertensive renomedullary lipids are vasodilators and it has been proposed that a deficiency of these lipids may contribute to the hypertension produced by destruction of the renal papilla. The aim of the present work was to study ACTH hypertension in both control and chemically renomedullectomized rats. Renomedullectomy was produced by single intraperitoneal injection of 2-bromoethylamine (BEA) at 400 mg/kg. BEA-treated rats all developed increases in water intake and urine volume, with loss of papillae and medullary and cortical fibrosis. There was a significant correlation between papillary ablation and systolic blood pressure (SBP). SBP in renomedullectomized rats was higher after ACTH than sham injection, and higher than after ACTH injection in intact rats. Chemical renomedullectomy with BEA did not block or attenuate the onset or magnitude of ACTH hypertension in the rat.


Extracellular calcium, contractile activity and membrane potential in tail arteries from genetically hypertensive rats. This study compares the effect of extracellular calcium on contractile responsiveness and membrane potential (E(m)) in arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive (WKY) rats. Isometric force and E(m) were measured in isolated tail artery strips using standard muscle bath and microelectrode techniques, respectively. The resting contractile force in SHRSP and WKY arteries was not influenced by the extracellular calcium concentration. However, the rate of force development in response to norepinephrine (3 x 10(-8) mol/l) was slowed when calcium was elevated and increased when calcium was reduced. Compared with WKY rats, this stabilizing action of calcium on contractions to norepinephrine was reduced in SHRSP. In 1.6 mmol/l calcium, resting E(m) in SHRSP did not differ from that in WKY rats. Calcium-free buffer caused depolarization in SHRSP and WKY rats. Reductions in calcium below physiological levels resulted in depolarization, whereas elevations in calcium caused hyperpolarization. Regardless of the calcium concentration, E(m) values in SHRSP did not differ from those in WKY rats. Norepinephrine (3 x 10(-8) mol/l) caused a depolarization in WKY rat and SHRSP arteries, and the magnitude of this depolarization was not influenced by calcium. Endothelium removal did not alter the stabilizing effects of calcium on the membrane potential or contractile activity in WKY rats or SHRSP. The reduced stabilizing effect of calcium on the contractile activity in SHRSP arteries is not due to an alteration in the general effect of the cation on the membrane potential.


Inhibition of vasopressin-induced formation of diradylglycerols in vascular smooth muscle cells by incorporation of eicosapentaenoic acid in membrane phospholipids. Eicosapentaenoic acid and linoleic acid exert antihypertensive effects by an unknown mechanism unrelated to prostanoids, a property which is not shared by arachidonic acid. This study investigated the influence of these three acids on the formation of diradylglycerols and phosphatidic acid, key intracellular messengers involved in the mediation of agonist-induced vascular smooth muscle cell contraction. Rat mesenteric artery vascular smooth muscle cells in culture were pre-incubated for 24 h with eicosapentaenoic acid, linoleic acid or arachidonic acid. After thorough washing the cells were then incubated for 20 min in the presence of arginine vasopressin or vehicle, either immediately or following cell labelling with 32P-orthophosphate. The fatty acid composition of cell lipids was determined by gas chromatography after transesterification in the presence of boron trifluoride and methanol. Diradylglycerols and 32P-phosphatidic acid were purified from cell lipid extracts by thin-layer chromatography and diradylglycerols were analysed. Incubation of vascular smooth muscle cells with eicosapentaenoic acid, linoleic acid or arachidonic acid resulted in the incorporation of these fatty acids at the sn-2 position of membrane phospholipids, mainly phosphatidylcholine and phosphatidylethanolamine. Eicosapentaenoic acid treatment was associated with a reduction, and linoleic acid treatment with an increase in the relative proportions of arachidonic acid found in cell phospholipids. Arginine vasopressin stimulated the formation of both diradylglycerols and 32P-phosphatidic acid. The arginine vasopressin-induced stimulation of diradylglycerols accumulation was almost completely abolished in eicosapentaenoic acid-treated cells, whereas it was not modified by linoleic acid or by arachidonic acid treatment. The arginine vasopressin-stimulated formation of 32P-phosphatidic acid was significantly inhibited by linoleic acid treatment but was not influenced by eicosapentaenoic acid or arachidonic acid treatment. The incorporation of eicosapentaenoic acid or linoleic acid at the sn-2 position of membrane phospholipids leads to an inhibition of arginine vasopressin-induced formation of diradylglycerols or phosphatidic acid, respectively, in rat mesenteric artery vascular smooth muscle cells in culture. These properties may contribute to the antihypertensive effects in these fatty acids in vitro.


The effect of angiotensin II on DNA synthesis varies considerably in vascular smooth muscle cells from different Wistar-Kyoto rats. It is well established that angiotensin II induces vascular smooth muscle cell (VSMC) growth but conflicting data exist concerning whether angiotensin II induces cell hypertrophy and/or DNA synthesis from Wistar-Kyoto (WKY) rats. In this study we examined the effect of 10(-7) mol/l angiotensin II on cell protein and DNA synthesis in VSMC (passages 5-30) derived from eight different WKY rats. The mitogenic and hypertrophic effect of angiotensin II was determined by 3H-thymidine incorporation into cell DNA and by total cellular protein measurements. In cells derived from two cell lines, angiotensin II induced a potent mitogenic effect. In cells derived from two other cell lines it induced a weak mitogenic effect. No significant effects on DNA synthesis were observed in cells derived from the four remaining cell lines. Cells from all cell lines reacted to angiotensin II with a 30-40% increase in cell protein. The observed mitogenic effect of angiotensin II was inhibited by losartan, a non-peptide angiotensin II receptor antagonist, proving that the angiotensin II-induced mitogenic effect is directly triggered via the angiotensin II subtype AT1 receptor. The hypertrophic effect of angiotensin II was also illustrated by morphological studies showing that angiotensin II increases the cell size in all cell lines used. Our study shows that angiotensin II not only is a hypertrophic agent, but may also be a potent mitogenic agent for VSMC from WKY rats.


Subpressor angiotensin II is a bifunctional growth factor of vascular muscle in rats. The proposition that angiotensin II in subpressor does stimulates vascular growth in vivo was tested. Young adult, male Sprague-Dawley rats received angiotensin II, 200 ng/kg per min intraperitoneally by osmotic minipump, for 24 h or 7-10 days. Sham-infused rats served as controls. Protein (35S-methionine) synthesis in aortic media, portal vein, bladder wall and diaphragm; proteoglycan (35S-sulfate) synthesis in aorta and bladder and synthesis of DNA (3H-thymidine) in aortic media were all measured ex vivo in the rat. The systolic blood pressure of angiotensin II-treated rats was unchanged at 24 h and increased at 7-10 days. At 24 h in angiotensin II-treated rats the protein synthesis in aortic media, portal vein and bladder wall but not in the diaphragm was increased, indicating that the hypertrophic effect of angiotensin II was independent of the arterial pressure. The rate of 35S-methionine washout from angiotensin II- and sham-treated aorta was the same. At 24 h there was also an increase in proteoglycans synthesis of the aorta and bladder wall of angiotensin II-treated rats. In contrast to protein synthesis, the incorporation of 3H-thymidine into aortic muscle DNA was reduced in angiotensin II-treated rats at 24 h, suggesting the inhibition of DNA synthesis. At 7-10 days angiotensin II administration the protein synthesis of aortic media returned to baseline, and DNA synthesis was bimodal: in 53% of rats (n = 10) inhibition continued, and in 26% (n = 5) it was increased by two- to threefold. The present findings confirm in vivo the bifunctionality of the trophic vascular action of angiotensin II. Vascular hypertrophy may play a role in the slow pressor action of angiotensin II.


Endothelin-1 stimulates tyrosine phosphorylation and the activities of two mitogen-activated protein kinases in cultured vascular smooth muscle cells. Endothelin-1 (ET-1) has been reported to stimulate the expression of the proto-oncogenes c-fos and c-myc, and to cause DNA synthesis in vascular smooth muscle cells (VSMC). The purpose of this study was to clarify the signalling pathway from ET receptors to the nucleus. Mitogen-activated protein (MAP) kinase, which is activated by various growth factors via phosphorylation of tyrosine and threonine residues, plays important roles as an intermediate in the signalling pathways from growth factor receptors to the ribosomes and nucleus. We examined the effect of ET-1 on the phosphorylation and activation of MAP kinase in cultured VSMC. Extracts of ET-1-stimulated VSMC were analysed by one- and two-dimensional gel electrophoresis and anion-exchange column chromatography. Tyrosine-phosphorylated proteins and MAP kinases were detected by immunoblot analyses with anti-phosphotyrosine and anti-MAP kinase antibodies, respectively. The MAP kinase activity was measured using myelin basic protein as a substrate. The MAP kinases were isolated from 32P-labelled VSMC and subjected to phosphoamino acid analysis. ET-1 induced tyrosine phosphorylation of at least five proteins of about 79, 77, 73, 45 and 40 kDa in VSMC. The mobilities of the tyrosine-phosphorylated 45- and 40-kDa proteins were identical with those of the two proteins that were recognized by anti-MAP kinase antibody upon one- and two-dimensional gel electrophoresis. ET-1 stimulated MAP kinase activity in a time-course similar to that of the tyrosine phosphorylation of the 45- and 40-kDa proteins. The ET-1-stimulated MAP kinase activity was resolved almost equally into two peaks upon Mono Q column chromatography (kinase 1 and kinase 2). Kinase 1 and kinase 2 were co-eluted with the tyrosine-phosphorylated 40- and 45-kDa proteins, respectively. The apparent molecular masses of kinase 1 and kinase 2 estimated by MAP kinase assay in polyacrylamide gel were identical with those of tyrosine-phosphorylated 40- and 45-kDa proteins, respectively. Upon phosphoamino acid analysis, ET-1 stimulated phosphorylation of MAP kinases not only on tyrosine but also on threonine residues. ET-1 induces tyrosine and threonine phosphorylation and the activation of two species of MAP kinases of 40 and 45 kDa in VSMC.


Morphometric study of cerebral arteries from spontaneously hypertensive and stroke-prone spontaneously hypertensive rats. The importance of sympathetic innervation for the development of structural changes in the cerebral arteries of hypertensive animals was studied. Sympathetic denervation was induced with combined treatment from birth of antibody against nerve growth factor and guanethidine. Previous studies from our laboratory showed that this procedure not only caused a permanent denervation of the mesenteric arteries, but also prevented the development of hypertension in spontaneously hypertensive rats (SHR). Morphometric measurement of the structural changes was carried out in the basilar, superior cerebellar, posterior cerebral and middle cerebral arteries from 28-week-old SHR, stroke-prone SHR, and normotensive Wistar-Kyoto rats. The results were compared with those obtained from cerebral arteries of sympathectomized rats. Total vascular wall cross-sectional area was significantly larger in the basilar and superior cerebellar arteries from hypertensive rats compared with normotensives. The change was characterized by an increase in the number of smooth muscle cell layers. There were also differences between the two hypertensive groups in some arteries. Sympathetic denervation attenuated the development of hypertension and vascular changes in some arteries. There was a positive linear correlation between blood pressure and medial cross-sectional area, and between blood pressure and the number of smooth muscle cell layers for the four arteries analysed. Sympathetic nerves have a trophic influence upon the remodelling of some cerebral arteries during the development of genetic hypertension.


Morphology and function of mesenteric resistance arteries in transgenic rats with low-renin hypertension. The renin-angiotensin system plays an important role in blood pressure control. Recently the mouse Ren-2 renin gene was introduced into the genome of rats, producing low-renin hypertensive animals. The aim of the present study was to characterize the pharmacological and morphological properties of mesenteric resistance arteries from transgenic rats. Segments of small arteries were taken from the mesenteric bed of 13-week-old transgenic rats and from age-matched Sprague-Dawley controls. Vessels were mounted on an isometric myograph permitting direct measurements of vessel isometric wall tension. Vessel morphology was measured with a microscope using a water-immersion objective. The transgenic versus Sprague-Dawley rat comparison is similar to that seen previously for spontaneous hypertensive rats (SHR) versus Wistar-Kyoto (WKY) rats as regards active effective pressure (increased), lumen diameter (decreased) and media thickness (increased). However, in contrast to SHR vessels, where media cross-sectional area has previously been shown to be increased compared with WKY vessels, vessels from transgenic rats had the same media cross-sectional area as those from Sprague-Dawley rats. There was neither cellular hypertrophy nor hyperplasia. However, an increased number of smooth muscle layers was found, indicating a rearrangement of existing material. Although the structural changes found in transgenic rats may account for the increased pressure response, hypertension in this animal is apparently not caused by general vascular growth.


Dietary gamma-linolenic acid lowers blood pressure and alters aortic reactivity and cholesterol metabolism in hypertension. To determine the effects of dietary gamma-linolenic acid upon blood pressure, aortic reactivity and cholesterol metabolism in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Randomized parallel-group study. SHR and WKY rats were fed a purified diet containing either sesame or borage oil rich in gamma-linolenic acid for 7 weeks. Blood pressure measured by the tail-cuff method and weight were monitored weekly. At the end of the study, intra-arterial pressor responses to norepinephrine and angiotensin II, and reactivity of isolated aortic rings to norepinephrine, angiotensin II, KCl and acetylcholine were determined. Serum cholesterol and triglycerides were measured. Hepatic and intestinal enzymes and receptors of cholesterol metabolism were also measured. Dietary borage oil significantly decreased blood pressure in SHR and WKY rats compared with sesame oil-fed rats. Pressor responses to norepinephrine and angiotensin II, and aortic reactivity to norepinephrine, angiotensin II, KCl and acetylcholine were not significantly different. The borage oil diet increased serum cholesterol levels in WKY rats and hepatic B-hydroxy-3-methylglutaryl coenzyme A reductase in SHR. These data indicate that dietary borage oil has a blood pressure lowering effect in hypertensive and normotensive rats. However, the effect cannot be explained by altered sensitivity to humoral and neural vasoconstrictors or changes in cholesterol metabolism. Other mechanisms should be investigated.


Erythrocyte membrane lipids and cationic transport systems in men. The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.


Ca2+ transport in unstimulated platelets of essential hypertensives and their blood relatives. Intracellular free Ca2+ concentration has been shown to be elevated in platelets from essential hypertensive patients. This study was designed to characterize Ca2+ homeostasis in platelets of essential hypertensives. A double-blind study was carried out. Untreated and treated (propranolol therapy) essential hypertensives were studied in comparison with normotensive control subjects. First-degree blood relatives of essential hypertensives were also studied. The various procedures used in the study were already standardized and well-established methods. For Ca2+ uptake and efflux studies, 45Ca was used. For intracellular free Ca2+ concentration studies the fluorescent Ca2+ chelator dye fura-2/acetoxymethyl ester (fura-2/AM) was used. The uptake of 45Ca by unstimulated platelets of untreated essential hypertensives and their relatives was significantly higher than for controls. However, essential hypertensives treated with a beta-blocker drug showed no significant difference in Ca2+ uptake compared with controls. A significantly decreased Ca2+ efflux was observed in essential hypertensives (both untreated and treated) compared with controls. Relatives also showed a depressed Ca2+ efflux compared with controls. It appears that the elevated intracellular free Ca2+ concentration levels in platelets (also observed by us) may be due to both an enhanced uptake into, and decreased efflux of Ca2+ from, the cell. Beta-blocker therapy may help to normalize the elevated intracellular free Ca2+ concentration levels observed in essential hypertensives. Relatives exhibit a state predisposed towards the development of hypertension.


Neuropeptide Y-like immunoreactivity and hypertension. Neuropeptide Y is a co-transmitter with noradrenaline in sympathetic neurons supplying arteries and veins with potent contractile effects. To investigate the role of neuropeptide Y in hypertension, we measured the circulating levels of neuropeptide Y and noradrenaline in patients with severe hypertension. Samples were collected from patients with untreated, severe hypertension (diastolic blood pressure > 120 mmHg) and in age- and sex-matched controls. After treatment with beta-adrenoceptor blockers, diuretics, angiotensin converting enzyme inhibitors of calcium antagonists, samples were taken from the patients during 12 months. The circulating levels of neuropeptide Y-like immunoreactivity (NPY-LI) were measured with a radioimmunoassay using a rabbit antiserum. Catecholamines were measured using high-performance liquid chromatography and electrochemical detection. There was a significantly higher level of NPY-LI in the patients when they were compared with the controls. However, there was no correlation either in the controls or in the hypertensives between systolic blood pressure, diastolic blood pressure and NPY-LI or noradrenaline. The increased level of NPY-LI in plasma remained elevated for up to 12 months despite reduction in blood pressure to acceptable levels. The noradrenaline level was not increased before treatment, after 2-4 weeks or after 2-12 months treatment. The high level of NPY-LI may represent a marker for higher activity of the sympathetic nervous system which is not controlled by the treatment of blood pressure to normotension.


Circadian rhythm of calcitonin gene-related peptide in uncomplicated essential hypertension. To assess the existence of an altered circulating pattern of calcitonin gene-related peptide (CGRP) in hypertension. The 24 h variation in plasma CGRP was measured and compared in 10 patients affected by uncomplicated essential hypertension and in nine age- and sex-matched healthy volunteers. The diurnal variations in blood pressure, atrial natriuretic peptide (ANP), plasma renin activity (PRA), plasma aldosterone and plasma cortisol were also assessed. Recumbency studies were performed under standardized, drug-free conditions beginning at 0800 h. Venous samples were drawn every 4 h for 24 h and hormone levels were assessed with specific radioimmunoassays. The blood pressure was measured every 15 min with a SpaceLabs 90207 monitor. The mean 24-h plasma CGRP concentrations were significantly lower in the hypertensive group than in the control group. In both groups a circadian rhythm was present with the same pattern, but at a lower level in hypertension. A temporal sequence starting with the nocturnal rise in plasma CGRP concentrations and progressing with the elevations of ANP, PRA, and plasma aldosterone and cortisol was apparent in both groups. The nocturnal rise in the CGRP and ANP concentrations coincided with the blood pressure and the heart rate falls. Our data show that CGRP is lower than normal but maintains its circadian variability and its relationship with the diurnal variations in blood pressure and other hormones known to be active on the cardiovascular system.


Cuff bladder width and blood pressure measurement in children and adolescents. To estimate the independent contribution of the blood pressure cuff bladder width: mid-upper arm circumference (CW:AC) ratio to the variability in systolic blood pressure (SBP), diastolic blood pressure phase 4 (DBP4) and diastolic blood pressure phase 5 (DBP5) in children and adolescents, and its impact upon the estimate of prevalence of high blood pressure derived at screening. Use of cuffs with a CW:AC ratio below 40% causes overestimation of the true blood pressure level. With higher CW:AC ratios the level is underestimated. National recommendations for adults state that the CW:AC ratio is optimal at approximately 40%, but no consistent recommendations exist for children. We measured SBP and diastolic blood pressure phases 4 and 5 in 811 boys and 771 girls aged 10-17 years (93% participation rate). In each subject the blood pressure was measured with a cuff conforming to the 40% rule (recommended cuff), with one smaller (mean decrease in the CW:AC ratio:7.4%), and with one larger (mean increase in the CW:AC ratio:10.2%). Cuffs were used in random order. Smaller cuffs gave significantly higher blood pressure readings. With the larger cuff there were significant mean decreases. The differences were independent of the blood pressure level obtained with the recommended cuff. The impact upon the estimate of the prevalence of high blood pressure was substantial. Selection of the proper cuff size is important for children and adolescents, in order to avoid both over- and underdiagnosis of hypertension.


Ambulatory blood pressure monitoring: a critical review of the current methods to handle outliers. To examine the methods to handle marginal readings in the analysis of ambulatory blood pressure. Data obtained from automatic ambulatory blood pressure monitoring include several 'outliers', i.e. readings at the frontier of physiologically acceptable ranges. Several methods have been used to handle these readings. We need to know whether using different methods to reject outliers leads to different results in the analysis of the data. If so, then it is important that a common method be used by different authors. Ten reported methods to handle outliers were selected and applied to a large set of unpublished blood pressure profiles (Novacor Diasys system). We compared the effects of data rejection by these methods on the mean values and standard deviations (calculated over 24 h, daytime and night-time) of the remaining data. The different methods had quite different effects on the same data set. Depending on the method used, the discarded data varied from 1 to 17% of the total number of readings. Among the rejected data, readings that occurred in the daytime varied from 14 to 56%. Also, 'high-value' outliers varied from 1 to 60% of the rejected data. On average over the total sample, the rejection of outliers had only a small effect on the mean values of blood pressure. In contrast, it may strongly reduce the standard deviation of the readings. The study emphasized the need to use a common method to handle outliers in the analysis of ambulatory blood pressure data.


Reproducibility of nurse-measured, exercise and ambulatory blood pressure and echocardiographic left ventricular mass in borderline hypertension. To compare the short-term reproducibility of four diagnostic tests: resting blood pressure, exercise blood pressure, non-invasive daytime ambulatory blood pressure and echocardiographic left ventricular mass. Blinded, prospective test-retest (reliability) study. Hypertension research units in two teaching hospitals. Six normal volunteers and 22 patients with untreated borderline to mild hypertension, mean age 44 years. The intraclass correlation coefficient (RI) and standard deviation of the difference (SDD) between visits. The mean blood pressures and left ventricular mass did not differ between visits. Concordance between visits reached RI = 0.86 systolic/0.66 diastolic for ambulatory blood pressure and RI = 0.85 systolic/0.64 diastolic for nurse-measured random-zero sphygmomanometer resting blood pressure. The respective variabilities were SDD = 9/8 and 8/8 mmHg. Submaximal exercise systolic blood pressure (SBP) and echo left ventricular mass showed excellent reliability. Echo left ventricular mass and resting SBP or ambulatory SBP were significantly more reproducible than resting diastolic blood pressure (DBP) or ambulatory DBP. Despite averaging many readings within each day, clinically important between-visit variations in ambulatory blood pressure remained. The between-visit variability of daytime ambulatory blood pressure was similar to that of resting blood pressure when carefully measured by a research nurse. The echo left ventricular mass appears to be more reproducible over the short term than the current diagnostic standard for hypertension, the resting DBP.


Left ventricular structure and determinants in normotensive offspring of essential hypertensive parents. To assess whether currently normotensive offspring of essential hypertensive parents may have alterations in left ventricular mass (LVM) and function, and how these relate to some potential determinants. Echocardiographical indices of LVM (assessed by two-dimensional guided M-mode echocardiogram), 'clinic' blood pressure and daytime ambulatory blood pressure profiles, blood pressure responses to dynamic and isometric exercise testing, haematocrit, plasma and 24-h urinary electrolytes and catecholamines, and plasma angiotensin II were assessed on a defined Na+ intake in 31 normotensive lean sons of essential hypertensive parents (OHYP group) and 30 body mass index- and age-matched sons of normotensive parents (ONORM group). Clinic supine systolic blood pressure was higher in the OHYP than the ONORM group, but clinic diastolic and daytime ambulatory mean blood pressures, blood pressure loads and blood pressure during dynamic or isometric exercise did not differ significantly. LVM index (LVMI), interventricular septum thickness (IVST), posterior wall thickness (PWT), the IVST:PWT ratio, ejection fraction, fractional shortening, cardiac index and measured biochemical variables also did not differ significantly between groups. In the whole study population the LVMI correlated positively with the body mass index and negatively with plasma noradrenaline. In young lean men with one essential hypertensive parent and blood pressure still in the normal range, left ventricular structure and systolic function, as assessed by echocardiography, seem to be often unaltered and appropriate relative to the existing body habitus and blood pressure. Moreover, an early tendency for increasing resting blood pressure in genetically hypertension-prone humans may be more apparent under clinic than usual ambulatory conditions, whereas the blood pressure reactivity to physical stress seems to be largely normal at this stage.


Prognosis in hypertensives with acute myocardial infarction. A previous history of hypertension is overrepresented among patients with ischaemic heart disease. The present study aims at describing the influence of a previous history of hypertension upon the prognosis among patients hospitalized due to acute myocardial infarction. Patients were followed for 1 year. Mortality and morbidity are described during hospitalization and after discharge from hospital. Sahlgrenska Hospital, serving half of the area of Gothenburg in Sweden. All patients admitted to Sahlgrenska Hospital during 21 months due to acute myocardial infarction regardless of age and whether they were admitted to the coronary care unit. Among all patients with confirmed acute myocardial infarction (n = 917) a previous history of hypertension was reported in 324 patients. Hypertensives more frequently had a previous history of acute myocardial infarction, angina pectoris, congestive heart failure and diabetes mellitus. Their mortality during hospitalization was similar to that in normotensives. However, the total mortality during 1 year of follow-up was 35% in hypertensives and 25% for normotensives (P < 0.01), and a previous history of hypertension was an independent risk indicator for death after discharge from hospital. Place and mode of death appeared similar in normotensives and hypertensives. Reinfarction was twice as common in hypertensives as in normotensives, and a previous history of hypertension was an independent risk indicator for reinfarction. Among patients with acute myocardial infarction a previous history of hypertension indicates a poor prognosis, one-third of patients dying and one-quarter developing reinfarction during the first year after onset of acute myocardial infarction.


Relation between treated blood pressure and death from ischaemic heart disease at different ages: a report from the Department of Health Hypertension Care Computing Project. To determine the relation between mortality from ischaemic heart disease (IHD) and treated blood pressure at different ages. Prospectively, 6216 patients were studied for a mean of 107 months. Of the total patients, 95% were followed in five hospital-based hypertension clinics and the remainder in four group general practices. Respectively, 2250 and 2126 hypertensive men and women aged < 60 years and 822 and 1018 aged > or = 60 years. Mortality (any mention on the death certificate) from IHD. Four hundred and sixty-seven patients died with IHD mentioned on the death certificate. The relation between both diastolic blood pressure (DBP) and systolic blood pressure (SBP) during the first 3-12 months of treatment and subsequent IHD mortality was examined. Under the age of 60 years the relative hazard rate (RHR) for death from IHD tended to increase with DBP in both men and women. Above the age of 60 years there was no important or significant relation between IHD mortality and treated DBP. For SBP there was no reduction in the positive relation between IHD mortality and blood pressure in the older age groups. The RHR for SBP ranged between 1.008 and 1.021 in men and women over and under the age of 60 years. The positive relation between DBP and IHD mortality decreased with increasing age and, in women aged > or = 60 years, even inverted, partly explaining the negative relation reported between DBP and total mortality in the very old.


Effects of mild physical activity, atenolol and the combination on ambulatory blood pressure in hypertensive subjects. To evaluate whether beta-blocker treatment could enhance the effect of a mild physical training programme upon blood pressure. In 12 hypertensive subjects (mean age: 40.3 years) a prospective randomized Latin square-design trial was performed with three treatments: physical training and placebo tablets; atenolol 50 mg once a day and inactivity; and physical training and atenolol 50 mg once a day. Training significantly increased maximal ventilatory oxygen consumption (VO2MAX), and there was a decrease in ambulatory diastolic blood pressure (DBP) which did not reach statistical significance. Atenolol alone significantly reduced ambulatory systolic blood pressure (SBP) and DBP. Atenolol alone did not reduce VO2MAX. The combination of training and atenolol resulted in an increase in VO2MAX compared with atenolol alone, but no additional significant fall in blood pressure. Atenolol did not enhance the effect of physical training upon blood pressure and had little if any effect upon the training-induced increase in exercise tolerance.


Expression of cellular retinoid-binding proteins during normal and abnormal epidermal differentiation. Retinoids have important roles in growth and differentiation of epidermal cells. We have analyzed the expression of two intracellular retinoid-binding proteins, the cellular retinol-binding protein type I and the cellular retinoic acid-binding protein type I, during normal and abnormal epidermal differentiation. Both proteins were found to be expressed in normal epidermis with increasing expression from basal layer towards superficial layers. In psoriatic lesions, a hyperproliferative condition of the skin, the epidermal expression of cellular retinol-binding protein I was induced, whereas expression of cellular retinoic acid-binding protein I was sharply down-regulated. This and other features of psoriatic lesions indicate that down-regulation of cellular retinoic acid-binding protein I expression might cause aberrant retinoid-regulated gene expression in skin. In basal and squamous cell carcinomas, cellular retinoic acid-binding protein I expression was down-regulated, whereas cellular retinol-binding protein I was expressed. Apart from epidermal cells, a mesenchymal, dendritic cell-type, strongly expressing cellular retinoic acid-binding protein I, was identified in the dermis. In several hyperproliferative conditions of the skin, including psoriasis, and squamous and basal cell carcinomas, this cell type was abundant. These results have implications for the role of retinoids in normal and abnormal epidermal differentiation and suggest that part of the phenotype of psoriasis is due to inappropriate metabolism of retinoic acid in skin.


Characterization of a partial cDNA for lysyl hydroxylase from human skin fibroblasts; lysyl hydroxylase mRNAs are regulated differently by minoxidil derivatives and hydralazine. Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that catalyzes the formation of hydroxylysine required for intermolecular crosslinking of collagen. We have isolated a partial (2.2-kb) cDNA for LH from human skin fibroblasts using PCR. DNA sequencing revealed 72% homology of the human coding sequence with the chick LH sequence at the nucleotide level and 76% homology predicted at the amino acid level. The LH cDNA hybridized strongly with two mRNA species of 2.4 and 3.4 kb on Northern blots of normal fibroblast RNA. Administration of minoxidil decreased both mRNA species without affecting levels of the mRNAs for the beta subunit of prolyl 4-hydroxylase (PH) or alpha 1(I) collagen. Two derivatives of minoxidil (3' hydroxyminoxidil and 4' hydroxyminoxidil) produced similar decreases in LH mRNAs. In contrast hydralazine increased the mRNAs for LH in parallel with its previously reported effect on the mRNA for the beta subunit of PH. This effect is accompanied by virtual elimination of the alpha 1(I) collagen mRNAs. These results on the action of minoxidil and hydralazine at the pretranslational level correlate well with their previously reported effect on enzyme activity and collagen biosynthesis and indicate that changes in steady-state mRNA levels can account directly for changes at the protein level. Moreover, the unique action of minoxidil in specifically decreasing LH mRNAs contrasts with the less specific stimulatory effects of hydralazine and suggests that these pharmaceuticals are regulating expression of LH at a pretranslational level by different mechanisms.


Effects of tetanus toxin, Salmonella typhimurium porin, and bacterial lipopolysaccharide on platelet aggregation. Endotoxins may interfere with platelet aggregation by interacting with the platelet membrane. The aim of this study was to evaluate the effects of tetanus toxin, Salmonella typhimurium porin, and bacterial lipopolysaccharide (LPS) on platelet aggregation induced by ADP and thrombin in vitro. Spontaneous platelet aggregation and platelet aggregation induced by ADP and thrombin were measured. Our results show that Salmonella typhimurium porin and bacterial LPS enhanced human and rabbit platelet aggregation induced by ADP and thrombin. Tetanus toxin did not affect platelet aggregation.


Extrachromosomal human immunodeficiency virus type 1 DNA forms in fresh peripheral blood lymphocytes and in two interleukin-2-independent T cell lines derived from peripheral blood lymphocytes of an asymptomatic seropositive subject. Two immature T cell lines (FT1 and FT4) were established after in vitro cloning of peripheral blood lymphocytes (PBLs) from an asymptomatic human immunodeficiency virus type 1 (HIV-1) seropositive, human T cell-lymphotropic virus type 1 seronegative homosexual subject. Although derived from a limiting dilution cell cloning assay, these cell lines were not recloned for this study. Their growth was independent of exogenous interleukin-2. Both cell lines were able to form colonies when cloned in agar, but failed to form solid tumours when injected into nude mice. FT lines belong to the very immature T cell lineage as they exhibit rearranged TCR genes but no expression of T cell membrane antigens, including CD2, CD3, CD4, CD6, CD7 and CD8. They also contain an HIV-1 genome that was detected only in an extra-chromosomal DNA form, even after several passages in vitro. The presence of unintegrated viral DNA was also detected by polymerase chain reaction analysis in the same sample of fresh uncultured PBLs. Furthermore, despite the absence of CD4 expression, both T cell lines were susceptible to CD4-independent HIV-1 superinfection (lack of superinfection inhibition in the presence of OKT4A monoclonal antibodies).


Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription. Vesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 micrograms/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with alpha- or gamma-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.


Expression of genes for the Epstein-Barr virus small RNAs EBER-1 and EBER-2 in Daudi Burkitt's lymphoma cells: effects of interferon treatment. The relative levels, rates of synthesis and stabilities of the abundant Epstein-Barr virus (EBV)-encoded small RNAs, EBER-1 and EBER-2, were examined in Daudi Burkitt's lymphoma cells. Although both RNAs are transcribed at approximately equal rates, the steady-state level of EBER-1 is at least 10-fold greater than that of EBER-2. This is shown to be due to a much faster rate of turnover of EBER-2. In the presence of actinomycin D, the half-lives of EBER-1 and EBER-2 are 8 to 9 h and 0.75 h, respectively. Following treatment of the cells with human interferon (IFN) alpha the transcription of both RNAs is strongly inhibited. However, the level of EBER-1 increases up to twofold, indicating a further stabilization of this RNA. In IFN-treated cells, EBER-2 accumulates in the form of truncated products. Nuclease protection experiments indicated that this is due to a post-transcriptional modification of the 3' end of the molecule. These data show that the effects of IFN treatment on the expression of these two viral gene products are very complex in cells latently infected with EBV.


On the structure, genesis and significance of DNA duplications at the Rous sarcoma proviral insertion sites in Rat-1 cells. We have previously shown that most Rous sarcoma proviruses in Rat-1 cells are simple insertions, at apparently random sites, and are not transcribed. A small minority of simple insertions are transcribed and these show a bias to insertion at sites closely 3' to presumptive cellular CG-rich islands. However, most transcribed proviruses are complex, typified by contiguous duplications of proviral DNA 5' to a complete proviral unit. The cellular sites of these complex insertions are unknown and their structure and significance incompletely documented. We report here more extensive analyses of proviral duplications, with the following findings. The 5' duplications predominantly involve proviral regions known to contain enhancer functions, substantiating earlier data. The cellular insertion sites are not biased to CG-rich loci at the level displayed by simple transcribed proviruses. The detailed structure of two duplications, and partial analysis of several others, strongly favours their genesis by illegitimate template transfer at reverse transcription, followed by self-recombination. These findings show that aberrant reverse transcription can generate duplications that dispense with the need for an expressed provirus to be integrated at a favourable cellular site.


The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker. Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.


Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types. DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.


In vitro infection of normal human keratinocytes by human papillomavirus type 1 followed by amplification of the viral genome in reconstructed epidermis. Primary cultures of normal human keratinocytes were inoculated in vitro with human papillomavirus type 1 (HPV-1), the agent responsible for deep plantar warts. Upon transfer to dead de-epidermized dermis and growth at the air-liquid interface, keratinocytes reconstituted a pseudoepidermis. Under these highly differentiating conditions, HPV-1 DNA amplification was found to take place in the reconstructed epidermis, being detectable from 7 days after the transfer and persisting for at least 10 days thereafter. The extent of keratinocyte differentiation may be insufficient to allow a complete HPV infectious cycle.


Primary chimpanzee skin fibroblast cells are fully permissive for human cytomegalovirus replication. Cytomegaloviruses generally display a host range restricted to differentiated cell types from the species they infect. For human cytomegalovirus (HCMV) this has meant that with few exceptions tissue culture systems have relied on the use of primary foreskin fibroblast (HF) cells or primary human embryonic lung cells to study gene expression and virus replication functions. We have observed that primary skin fibroblast (CF) cells derived from the chimpanzee (Pan troglodytes) support the replication of a laboratory strain (Towne) of HCMV. The kinetics of gene expression of the Towne strain grown in CF or HF cells appeared to be equivalent. Titres of progeny virions grown in CF cells appeared to be reduced 10-fold relative to those of virus grown in HF cells. In contrast, replication of the Towne virus was not supported by growth in WES cells (ATCC no. CRL 1609), a chimpanzee skin fibroblast cell line transformed by an adenovirus 12-simian virus 40 hybrid. This study shows that HCMV is less parochial in its host range than previously thought.


Differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin D. The growth of feline enteric coronavirus strain 79-1683 in whole feline embryo cells was inhibited by the presence of 1 microgram/ml of actinomycin D in the culture fluid. No virus-specific mRNAs could be detected in such cultures and yields of infectious virus were depressed by > 99%. By contrast, the antigenically related feline infectious peritonitis virus strain 79-1146 was unaffected by the presence of actinomycin D, indicating a fundamental difference between the two feline coronavirus strains in their requirements for host-encoded function(s).


Extensive antigenic diversification of foot-and-mouth disease virus by amino acid substitutions outside the major antigenic site. The antigenic sites A and C (the G-H loop and the C terminus, respectively) in VP1 of foot-and-mouth disease virus (FMDV) have been considered the immunodominant regions of the virus involved in the induction of protection. Other antigenic sites have been described but their involvement in protection has not been established. Here we report that two closely related but serologically different FMDVs (the field isolate C3 Argentina/84 and the vaccine strain C3 Resende Br/55) have identical A and C sites but differ as other antigenic sites. Such differences have been documented by reactivity with a panel of 28 monoclonal antibodies (MAbs). The two viruses reacted to the same extent with each of 13 MAbs which recognized epitopes within sites A or C, but reacted differently with six out of 15 MAbs that recognized other sites. Accordingly, sequencing of the entire region coding for the capsid proteins, for both viruses, revealed four amino acid substitutions at three antigenic sites other than A and C. The results suggest that identity of sites A and C may not be sufficient to induce cross-protection, and provide the first evidence of significant antigenic diversification of FMDV in the field mediated by amino acid substitutions outside sites A or C.


Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons. Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited > or = 80%. When protein synthesis was inhibited within 22 +/- 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 +/- 4 h. Analogously, the commitment point for RNA synthesis was 26 +/- 4 h and that for NGF rescue, 24 +/- 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGF beta-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death.


Hepatoma presenting as craniospinal metastasis: analysis of sixteen cases. Sixteen cases of hepatoma presenting as craniospinal metastasis without obvious hepatic involvement were reviewed. Metastatic spread of hepatoma to the cerebrum was found in one case, to the cranium in six cases, and to the vertebrae in nine cases. All of these cases had midly abnormal liver function when first evaluated. Of those patients with hepatoma, 56% had evidence of hepatomegaly. Alpha fetoprotein was present, at levels greater than 320 ng/ml, in 69%. Ninety four per cent died of the primary liver disease within one year. For any patient who develops craniospinal metastasis of unknown origin in a geographical area where hepatoma is a common disease, hepatoma should be considered in the differential diagnosis.


Occurrence of polyglucosan bodies in temporal lobe epilepsy. Massive occurrence of polyglucosan bodies (PBs) was found within the surgically removed temporal lobe of a 34 year old woman with complex partial seizures. This peculiar feature is very unusual in neuropathological examinations of epileptogenic foci. This patient could not be included in any of the classic diseases in which PBs are found. She exhibited a localised form of glycogen storage disease.


The environment in childhood and risk of motor neuron disease. To investigate a possible link between subclinical infection with poliovirus in childhood and increased risk of motor neuron disease in adult life, environmental determinants of infection in early life were compared in 98 cases of motor neuron disease and 335 age and sex matched controls. A weak but consistent relation was found between motor neuron disease and factors in the childhood environment known to increase likelihood of enteric infection. Relative risks associated with spending the first 10 years of life in a house without domestic amenities such as a bathroom, running hot water or flushing lavatory, living in overcrowded conditions, frequent changes of address or having a sibling with paralytic poliomyelitis were all greater than unity, although only those for absence of running hot water and frequent changes of address were statistically significant.


Quantitative morphometric study of muscle in inclusion body myositis. Clinical and electromyographic findings do not clearly distinguish inclusion body myositis (IBM) from chronic polymyositis (PM). The rimmed vacuoles and filamentous nuclear and cytoplasmic inclusions that characterize IBM are often sparse and may be overlooked; conversely, these features may occasionally be seen in other diseases. Preliminary studies suggested that muscle fiber hypertrophy occurred more frequently in IBM than in PM. To investigate whether fiber hypertrophy can be used to improve the ability to separate IBM from PM, we report a morphometric analysis of 28 IBM cases, 22 PM and 22 dermatomyositis (DM) cases. The analysis, using a computer automated system, included proportion of hypertrophied fibers and also fiber type proportions, average fiber diameter, proportion of atrophic and angulated fibers, and the co-dispersion index (CDI). The proportion of hypertrophied fibers was greater in IBM than the other two conditions (IBM (mean +/- SEM) 31.0 +/- 4.7% and 12.2 +/- 2.4% for type 1 and type 2 fibers, respectively, compared to 9.8 +/- 3.0% and 3.3 +/- 1.7% in PM, and 7.7 +/- 2.7% and 3.9 +/- 1.9% in DM). These differences were statistically significant (P < 0.05) in both sexes for type 1 fibers and in women for type 2 fibers. Also, the average fiber size and hypertrophy factors for type 1 and type 2 fibers were increased in IBM compared to PM and DM. This study confirms that the presence of muscle fiber hypertrophy in biopsies from IBM patients may help differentiate them from other clinically similar inflammatory myopathies.


Ubiquitin in cerebral amyloid angiopathy. Immunohistological findings in cerebral blood vessels of 4 cases with cerebral amyloid angiopathy (CAA) were compared with those of 4 Alzheimer's (AD) cases. A panel of antibodies against 2 neurofilament subunits (BF10 and RT97), a microtubule-associated protein (TAU) and ubiquitin were used. CAA cases showed a strong immunoreactivity for ubiquitin in blood vessel wall. Senile plaques (SPs) in CAA cases showed strong ubiquitin positivity but the central amyloid core was negative. AD brains showed immunoreactivity with all antibodies in SPs and neurofibrillary tangles (NFTs); blood vessels were consistently negative for ubiquitin. Control brains showed few SPs and NFTs; these were positive for ubiquitin, but blood vessels were negative. These results indicate that vascular amyloid deposition in CAA and AD may have different pathophysiological mechanisms.


Amyloid polyneuropathy with transthyretin Arg50 in a Japanese case from Osaka. A Japanese patient with systemic amyloidosis associated with a transthyretin (TTR) variant Arg50 is presented. This 41-year-old man became impotent and developed decreased pain sensation in his hands, and then sensory loss and muscle wasting in his lower legs, and cardiomyopathy appeared. The symptoms progressed and he died of congestive heart failure at age 46. There were amyloid deposits in all organs studied and massive amyloid deposition was seen in the peripheral nerves and cardiac muscles. Amyloid fibrils extracted from heart tissue contained TTR. A genetic mutation, causing a Ser50-->Arg substitution of the TTR molecule, was identified in another family member. Plasma TTR was shown to be a mixture of normal TTR Ser50 and mutant TTR Arg50 in the 2 subjects.


Role of calcium in inactivation of calcium/calmodulin dependent protein kinase II after cerebral ischemia. Transient cerebral ischemia demonstrates an increase in activated oxygen species in the brain that could lead to eventual neuronal cell death. Neuronal cells respond to oxygen free radicals through the restructuring of the cytoskeleton and membranes, mobilization of calcium and gene expression which play a role in cell injury. Ten min of bilateral carotid artery occlusion resulted in a decrease in calcium/calmodulin dependent protein kinase II (CaM kinase II) phosphorylation and activity detected in the brain immediately following ischemia and was partially restored within 24 h of reperfusion. Pretreatment of animals with an anesthetic dose of pentobarbital (40 mg/kg) resulted in partial protection of inactivation of CaM kinase II following ischemia. CaM kinase II activity was maintained following pretreatment of animals with alpha-phenyl N-tert-butyl nitrone (PBN), which traps oxygen free radicals. Infusion of superoxide dismutase or catalase prior to ischemia, blocked CaM kinase II inactivation. Blockage of calcium uptake with bepridil resulted in a marked protection of CaM kinase II inactivation. In addition, trifluoperazine, a calmodulin antagonist also diminished the inhibition of CaM kinase II phosphorylation in our model. These results suggest that ischemia and reperfusion injury results in the generation of activated oxygen and the mobilization of calcium which inactivate CaM kinase II. These results indicate that changes associated with protein kinase activity in the brain following an ischemic insult may have profound effects upon neurodegeneration and neuronal survival.


What do we really know about amyotrophic lateral sclerosis? The cause of amyotrophic lateral sclerosis is unknown. In this review clinical and scientific data that are pertinent to understanding this disease are reviewed. There are currently several major controversies concerning the possible role of immunological factors, genetic factors, environmental toxins, and viral infection in pathogenesis. These concepts must be considered in relation to what is known about the disease in all its aspects, including epidemiological data, information on the classical and molecular pathology of the disease, and on associated involvement of other systems, e.g., the spinocerebellar pathways and frontal dementia. Only when all this information is assimilated can full understanding of the disease and, hopefully, a logical approach to treatment and prevention, be achieved.


Comparative localization of corticotropin and corticotropin releasing factor-like peptides in the brain and hypophysis of a primitive vertebrate, the sturgeon Acipenser ruthenus L. The sturgeon is a primitive actinopterigian fish that, unlike modern teleosts, possess a portal vascular system that connects a true median eminence with the anterior pituitary as in mammals. The occurrence and localization of corticotropin and corticotropin releasing factor-like immunoreactivies were examined in the brain of the sturgeon (Acipenser ruthenus L.) by immunocytochemistry with antisera raised against synthetic non-conjugated human corticotropin, and rat/human corticotropin releasing factor. In the hypothalamus, corticotropin-immunoreactive parvicellular perikarya were found in the infundibular nucleus and in dendritic projections to the infundibular recess. In addition, ependymofugal corticotropin-immunoreactive fibres were found to terminate in the ventral hypothalamus. Corticotropin releasing factor-immunoreactive neurons were found in the rostral portion of the ventral hypothalamus (tuberal nucleus), and in the vicinity of the rostral aspect of the lateral recess. These cells projected to the dorsal hypothalamus, the ventral hypothalamus, the median eminence, the anterior and posterior telencephalon, the tegmentum mesencephali, and the pars nervosa of the pituitary. An affinity-purified UI antiserum failed to stain the sturgeon hypothalamus. Corticotrophs in the rostral pars distalis of the pituitary were also corticotropin-immunoreactive. In the neurointermediate lobe, only about 50% of cells of the pars intermedia appeared to be corticotropin-positive, the rest appeared unstained. These results suggest that the presence of corticotropin-like and corticotropin releasing factor-like peptides in the brain is a relatively early event in vertebrate evolution, already occurring in Chondrostean/Actinopterigian fishes, as exemplified by A. ruthenus. The close spatial relationship between corticotropin releasing factor immunoreactivity and corticotropin immunoreactivity in the ventral hypothalamus of A. ruthenus supports a possible interaction between the two systems in that area of the sturgeon brain. The pars intermedia might be an important site for corticotropin synthesis, even though the possibility cannot be excluded that the antiserum was recognizing the proopiomelanocortin molecule. The occurrence of corticotropin releasing factor immunoreactivity in the region of median eminence/pars intermedia of the sturgeon suggests that the sturgeon corticotropin releasing factor might regulate the adenohypophyseal release of proopiomelanocortin products in the same manner as in other vertebrates. The presence of extrahypothalamic corticotropin releasing factor-immunoreactive projections suggests further neuromodulatory functions for this peptide in A. ruthenus.


Suppression of high affinity IL-2 receptors on mitogen activated lymphocytes by glioma-derived suppressor factor. Previously we have reported that human glial tumor cells secrete a factor(s) which suppresses the mitogen responsiveness of normal human peripheral blood lymphocytes (PBL) in a dose dependent manner. In this study we extend these observations and explore the possible mechanisms by which glioma-derived suppressor factor(s) (GSF) modulates lymphocyte reactivity. Preincubation of lymphocytes with GSF for 2 hrs induces suppression of lymphocyte mitogen responsiveness. GSF also inhibits production of interleukin-2 (IL-2) by mitogen activated human T-cells. Addition of delectinated or recombinant IL-2 to mitogen activated human T-cells in the presence of GSF does not restore the normal proliferative response of these cells. These findings suggest that GSF induces a defect in the expression of the receptor for IL-2 (IL-2R) on activated T-cells. Binding studies with radiolabeled IL-2 demonstrated that GSF suppresses and in some cases completely inhibits the expression of functional high affinity IL-2R on activated T-cells, thereby, preventing association of IL-2R with its receptor and the subsequent progression of the cell into the proliferative stage of the cell cycle. These cellular defects induced by GSF closely parallel the observed defects noted in T-cells obtained from patients with gliomas, indicating that the factors elicited from glial tumors may be responsible for the immunological deficits observed in patients with primary malignant intracranial tumors.


Test for chemotherapeutic sensitivity of cerebral gliomas: use of colorimetric MTT assay. This study was undertaken to evaluate the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay as a means of testing the sensitivity of gliomas to chemotherapeutic agents in vitro. Eight human glioma established cell lines were plated in 96-well tissue culture plates and incubated for 4 days with 10 different anti-cancer agents; 5 different concentrations of each drug were tested. The MTT dye was then added to the wells, and the resulting formazan precipitate was solubilized with dimethylsulfoxide (DMSO). The spectrophotometric absorbance (measured at 570 nm) of control and experimental wells was used to calculate the cytotoxicity index (CI). Values with a CI greater than 50% growth inhibition indicated cytotoxic efficacy (sensitivity to the chemotherapeutic drug). Six of the seven (85.7%) glioma cell lines were highly sensitive at varying concentrations to mitomycin C, cisplatin, and doxorubicin. Four of the seven (57.1%) cell lines demonstrated intermediate sensitivity to mitoxantrone and vinblastine. Five of the seven (71.4%) cell lines exhibited resistance to etoposide, bleomycin, cosmegen, and BCNU. One of the cell lines tested, U-138MG, failed to produce the MTT formazan precipitate, so that the sensitivity of this cell line to the panel chemotherapeutic drugs could not be determined. The variability of the results indicates the need for an in vitro screening method to evaluate the effectiveness of clinical and experimental chemotherapeutic agents. The MTT assay provides a rapid method of screening antineoplastic agents against gliomas for cytotoxicity.


Increasing radiation dose intensity using hyperfractionation in patients with malignant glioma. Final report of a prospective phase I-II dose response study. We attempted to show a dose effect relationship for radiation therapy by treating patients harbouring malignant glioma with increasing doses of radiation in a step-wise fashion. We postulated that no increase in delayed toxicity would be seen because we used hyperfractionation technique. Between January 1981 and December 1988 we treated 280 patients three times daily at 4 hour intervals. 100 patients received a total dose of 6141 cGy, 73 patients received 7120 cGy, and 107 patients received 8000 cGy. CCNU was given at the time of tumor progression following radiotherapy. Median time to tumor progression was 28 weeks for patients who received 6141 cGy, 27 weeks for patients who received 7120 cGy and 36 weeks for patients who received 8000 cGy. Median survival was 46 weeks for patients who received 6141 cGy, 38 weeks for patients who received 7120 cGy and 45 weeks for patients who received 8000 cGy. There was no statistically significant difference in either time to tumor progression or survival among the three treatment arms and no dose response effect was seen. There was no increase in delayed radiation toxicity when the total radiation dose was increased up to 8000 cGy.


Detection of capsid antigen of human papillomavirus (HPV) in benign lesions of female genital tract using anti-HPV monoclonal antibody. We established a murine monoclonal antibody (K1H8) to human papillomavirus (HPV) using alkaline-disrupted virions of HPV type 1 (HPV-1) as the immunogen. K1H8 recognized a 57 kD capsid protein of HPV-1 and detected the antigen in paraffin sections of formalin-fixed tissue. With K1H8, we examined immunohistochemically 68 biopsy specimens obtained from the female genital tract. The specimens were histologically condyloma acuminatum or koilocytotic lesions with or without dysplasia and each specimen was found to harbour a single type of genital HPV, such as types 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58, by Southern blot hybridization analysis. The antigen was localized in the nuclei and occasionally in the cytoplasm of squamous cells showing koilocytotic changes. Eighty-four per cent of the specimens (57 cases) showed positivity for the antigen, indicating that K1H8 is a broadly-reactive antibody to various genital HPVs. The results suggest that benign mucosal lesions of the female genital tract are more frequently associated with viral production and are a potential source of transmission.


High incidence of EBV genome in CD30-positive non-Hodgkin's lymphomas. In Hodgkin's disease, Epstein-Barr virus (EBV) is found in CD30-positive Reed-Sternberg cells. We therefore studied 60 CD30-positive non-Hodgkin's lymphomas (NHLs) for the presence of EBV by the polymerase chain reaction (PCR) and DNA in situ hybridization (DISH), and by immunohistochemistry for the latent EBV proteins LMP and EBNA-2. CD30-negative NHLs and reactive lymph nodes served as controls. The CD30-positive cases comprised 17 anaplastic large cell lymphomas (ALCLs) (> 75 per cent CD30-positive cells) and 43 non-ALCLs (with 5-35 per cent CD30-positive cells). By PCR, 40 of 60 CD30-positive NHLs (67 per cent) were EBV-positive; in CD30-negative cases, 6/29 (21 per cent) were EBV-positive, as were 12/50 (24 per cent) reactive lymph nodes. The DISH procedure demonstrated the EBV genome exclusively in the nuclei of tumour cells in 23 of the 37 PCR EBV-positive cases that were tested. PCR-negative cases were always DISH-negative, as were the PCR-positive reactive lymph nodes and CD30-negative NHLs. Immunohistochemistry demonstrated LMP in neoplastic cells of 7/47 (15 per cent) CD30-positive NHLs, both ALCL and non-ALCL always in PCR EBV-positive cases, but never in the two control groups. EBNA-2 staining could not be detected. It is concluded that EBV is present (and transcriptionally active) in a sizeable number of NHLs and an association between the presence of the EBV genome and CD30 expression seems likely.


The detection of occult renal tumors in children by elevated hematocrit on routine complete blood count: a report of two cases. Wilms' tumor is a renal neoplasm usually found in young children and is rarely seen in teenagers. The production of erythropoietin by these tumors may result in secondary erythrocytosis, which should be reflected in complete blood counts (CBC). A search of the literature for reports of occult Wilms' tumors initially suspected on the basis of erythrocytosis was unrewarding. Two teenagers are reported in whom unexplained erythrocytosis was the initial indication of a Wilms' tumor. In both cases, a previous CBC showed elevations in hemoglobin and hematocrit that might have led to an earlier diagnosis. We conclude that unexplained erythrocytosis should sound the alert for further diagnostic studies to evaluate the possibility of occult renal neoplasia in older children.


Specific inhibition of leukotriene B4-induced neutrophil activation by LY223982. LY223982, (E)-5-(3-carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5- hexenyl)oxy)benzenepropanoic acid, is a newly discovered potent inhibitor of specific binding of leukotriene B4 (LTB4) to its receptor on human neutrophils. This study demonstrated that the compound is also a specific antagonist of LTB4-induced neutrophil activation under both in vitro and in vivo conditions. LY223982 was found to be 189-fold more effective in displacing [3H]LTB4 than 35S-N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) from their corresponding receptors on human neutrophils. The concentration inhibiting 50% of response (IC50) for displacement of [3H]LTB4 (13.2 nM) was only 6.8-fold higher than the value for nonradioactive LTB4. The compound inhibited the aggregation of guinea pig neutrophils caused by LTB4 more strongly than FMLP or platelet-activating factor. The IC50 for inhibition of LTB4-induced responses (74 nM) was 93- and > 135-fold lower than the IC50 for inhibition of the corresponding FMLP and platelet-activating factor-induced effects. LY223982 was also a potent antagonist of the aggregation of human neutrophils by LTB4 (IC50, 100 nM). Chemotaxis of human neutrophils induced by LTB4 was only modestly inhibited by the compound (IC50 = 6 microM) but it had even less effect on cell movement caused by FMLP. LY223982 inhibited transient leukopenia induced in rabbits with LTB4 (ED50, 3 mg/kg) but not with FMLP. It had no agonist activity in any of the test systems. In summary, the results indicate that LY223982 is a potent specific antagonist of LTB4-induced neutrophil activation.


D-16949 (anpirtoline): a novel serotonergic (5-HT1B) psychotherapeutic agent assessed by its discriminative effects in the rat. D-16949 [6-chlor-2-(piperidyl-4-thio)-pyridine; Anpirtoline] is a novel centrally acting compound with serotonergic effects. To assess its discriminative stimulus effects, rats were trained to discriminate D-16949 (2.0 mg/kg i.p., 30 min) from no drug. D-16949 induced dose-dependent discriminative stimulus effects (ED50, 0.31 mg/kg), and did not produce sedation. The opioid analgesics codeine, pentazocine and tramadol all failed to substitute for D-16949. The opioid antagonist naltrexone did not antagonize the discriminative stimulus effects of D-16949. Phencyclidine, d-amphetamine, lysergic acid diethylamide and quipazine produced between 0 and 35% responding on the D-16949 lever. 8-Hydroxy-2-(di-n-propylamino)-tetralin substituted partially (45%) for D-16949, whereas 1-(m-trifluoromethylphenyl)-piperazine and RU 24969 completely and dose-dependently substituted for D-16949. The discriminative stimulus effects of D-16949 were not reversed by either cyproheptadine, ketanserin, pirenperone, spiperone or methylsergide. The 5-hydroxytryptamine3 (5-HT3) active antagonists ICS 205-930 and MDL 72222 were also ineffective as D-16949 antagonists. It is concluded that the discriminative stimulus effects of D-16949 are not mediated through opioid or 5-HT2 mechanisms. The present data also do not suggest the involvement of 5-HT3 mechanisms, but that D-16949 produces its discriminative stimulus effects in the rat primarily via agonistic actions at 5-HT1B receptors.


Renal and systemic effects of aminoacids administered separately: comparison between L-arginine and non-nitric oxide donor aminoacids. The present study examined the mechanisms of the renal effect of the NO-donor aminoacid, L-Arg and different non-NO-donor aminoacids, namely L-Asn, L-Ala, L-Gly L-Gln administered separately. In conscious, unrestricted Wistar rats, a bolus of L-Arg produced a short-lasting decrease in mean arterial pressure. No variations in mean arterial pressure were found with either L-Gly, L-Asn, L-Ala or L-Gln. This effect of L-Arg was inhibited by NwNLA, methylene blue and atropine and not affected by meclofenamate. Simultaneously, a dose-response diuretic and natriuretic effect was observed with all the aminoacids. In further experiments with L-Arg and L-Gly, this effect was associated with increased glomerular filtration rate, renal plasma flow, fractional sodium and free water excretion and urinary cyclic guanosine monophosphate. These effects of L-Arg and L-Gly were inhibited by NwNLA. On the contrary, no inhibition by NwNLA was detected on the diuretic, natriuretic and renal hemodynamic effects of L-Gln, and the diuretic and natriuretic effects of L-Asn or L-Ala. Our results show that all the assayed aminoacids were endowed of diuretic and natriuretic capabilities. Such effects were apparently related with a NO-mediated mechanism in the case of L-Arg and L-Gly, but not in the case of L-Gln, L-Asn or L-Ala, therefore suggesting that more than one mechanism is involved in the renal effect of the different aminoacids. Simultaneously, only L-Arg produced a NO-, cyclic guanosine monophosphate-dependent hypotensive effect, which was not shared by the other assayed aminoacids.


Thermodynamic analysis of the temperature dependence of the dissociation constant of naloxone at opioid delta receptors in the mouse isolated vas deferens. Dissociation constants (KB) for naloxone inhibition of the actions of DPDPE in the mouse isolated vas deferens preparation (inhibition of electrically induced twitch) were determined at five temperatures ranging between 25 and 40 degrees C. The values of KB tended to increase with temperature over the range examined, indicating that the affinity of naloxone for the opioid delta receptor is an inverse function of temperature. Using these data, the thermodynamic quantities delta G zero' (change in free energy), delta H zero' (change in enthalpy) and delta S zero' (change in entropy) were calculated from a van't Hoff plot of in (KB) against 1/T. The thermodynamic quantities determined in this study in vivo (delta G zero' = -10.59 kcal mol-1, delta H zero' = -15.73 kcal mol-1 and delta S zero' = -0.0168 kcal mol-1 zero K-1) are consistent with data reported from radioligand binding studies in vitro and suggest that the interaction between naloxone and the opioid delta receptor in the mouse isolated vas deferens is enthalpy driven. These data represent the first evaluation of the thermodynamics of opioid antagonist/receptor interaction in a physiological assay.


Modulation by intracellular calcium pool of arginine vasopressin-induced cellular cyclic AMP production in rat renal papillary collecting tubule cells in culture. The present study was undertaken to determine whether endoplasmic reticulum is involved in the cellular action of arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture, using three dissimilar agents. 1 x 10(-7) M AVP increased cytosolic free Ca++ concentration ([Ca++]i) from 93.2 to 188.6 nM (P < .01). Exposure to 1 x 10(-4) M 3, 4, 5-trimethoxybenzoic acid, 8-(diethylamino) octylester hydrochloride (TMB-8), which inhibits intracellular Ca++ mobilization and blocks the function of endoplasmic reticulum, attenuated the [Ca++]i response to AVP. A significant increase in [Ca++]i in response to 1 x 10(-7) M AVP was obtained with Ca(++)-free medium containing 1 x 10(-4) M ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) (52.3 to 98.3 nM). However, when cells were preincubated with Ca(++)-free medium containing a mixture of 1 x 10(-4) M EGTA and 1 x 10(-4) M TMB-8, the 1 x 10(-7) M AVP-mobilized [Ca++]i was completely abolished. In the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine, AVP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner. Such an AVP-induced cAMP production was significantly reduced in cells exposed to Ca(++)-free medium containing 1 x 10(-4) M EGTA. After exposure to Ca(++)-free medium containing a mixture of 1 x 10(-4) M EGTA and 1 x 10(-4) M TMB-8, the cAMP response to AVP was markedly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)