Axoplasmic transport of [3H]ouabain binding sites and catecholamine secretion from an adrenergic nerve trunk. The presence of a functional Na+/Ca2+ exchange system was explored in the ligated cat hypogastric nerve, a preparation that has been proposed as a model of giant noradrenergic nerve terminal free of effector cells. The rationale for this study was to monitor noradrenaline secretion from the ligated cat hypogastric nerve promoted by the increase in intracellular Ca2+ levels after ouabain blockade of Na+,K(+)-ATPase molecules present in the plasma membrane of the ligated cat hypogastric nerve. Such an increase in intracellular Ca2+ levels is achieved by activation, in "reverse mode," of the Na+/Ca2+ exchange system. In the present study, [3H]ouabain binding sites were identified on crude preparations of hypogastric nerve membranes. A single, high affinity (Kd around 10 nM), binding site was observed in both ligated and nonligated nerves. The number of binding sites increased with the time of ligation, reaching a peak of about 1 pmol/mg of protein 48 hr after ligation. Blockade of these binding sites by ouabain induced a dose-dependent, Ca(2+)-dependent release of noradrenaline, with an ED50 around 50 microM. The maximum release amounted to 9% of the total noradrenaline content in the cells. As would be expected for ouabain-induced noradrenaline secretion mediated by a Na+/Ca2+ exchange system working in reverse mode, the effect of ouabain was dependent upon the presence of Na+ in the incubation medium, reaching a plateau at an extracellular Na+ concentration of 100 mM. Calcium uptake after Ca2+ reintroduction in ouabain-treated nerves increased with time of ligation, suggesting the incorporation of Na+/Ca2+ exchange carrier molecules into the axolemma of hypogastric nerves. The similarity between ouabain-induced noradrenaline secretion from the ligated cat hypogastric nerve and from other adrenergic systems strongly supports the idea that the ligated cat hypogastric nerve is equipped with a functional Na+/Ca2+ exchange system that would contribute to the regulation of intracellular Ca2+ levels. Furthermore, these data, together with previously published reports, fully characterize, from a biochemical point of view, the ligated hypogastric nerve as a model of giant noradrenergic nerve terminal free of effector cells.


Selective interaction of beta 2- and alpha 2-adrenergic receptors with stimulatory and inhibitory guanine nucleotide-binding proteins. In Chinese hamster ovary cells expressing recombinant beta 2-adrenergic receptors, isoproterenol enhanced cholera toxin-catalyzed ADP-ribosylation of the large form of G5 alpha. The effect was stereoselectively blocked by the enantiomers of propranolol, indicating receptor mediation. The ADP-ribosylated form of Gs alpha-subunit was resolved into a triplet in gradient gels. beta 2-Adrenergic receptors increased both the labelling and the apparent mass of the slower migrating forms of large Gs alpha, as determined by autoradiography and immunoblotting, suggesting that Gs alpha, can incorporate more than one ADP-ribose per molecule. In cells coexpressing similar amounts of beta 2-adrenergic, alpha 2-adrenergic, and m1 muscarinic receptors, beta 2 receptors stimulated the ADP-ribosylation of only large Gs and alpha 2 receptors that of only Gi; muscarinic receptors had no apparent effect. Thus, in native membranes there appears to be a selectivity for the interaction between adrenergic receptor subtypes and Gs alpha or Gi alpha subunits.


Opioid receptor-mediated inhibition of 3H-dopamine and 14C-acetylcholine release from rat nucleus accumbens slices. A study on the possible involvement of K+ channels and adenylate cyclase. The release of 14C-ACh from rat nucleus accumbens slices, induced by 15 mM [K+], was inhibited by the mu- and delta-opioid agonists DAMGO and DPDPE, respectively, whereas only the kappa agonist U50,488 reduced the release of 3H-DA. The opioid receptors involved appear to be localized on nerve terminals, since blockade of action potential propagation by 1 microM TTX did not diminish the inhibitory effects of DAMGO, DPDPE or U50,488. Enhancement of the potassium concentration in the superfusion medium to 56 mM with simultaneous reduction of the Ca2+ concentration from 1.2 mM to 0.12 mM induced a release similar to that caused by 15 mM K+ and 1.2 mM Ca+. Under this conditions, the inhibitory effects of both DAMGO and DPDPE on stimulated 14C-ACh release were reduced, whereas the inhibition of evoked 3H-DA release caused by U50,488 was not affected. Activation of mu- as well as delta-opioid receptors by DAMGO and DPDPE, respectively, inhibited forskolin-stimulated adenylate cyclase activity. However, increasing the intracellular cAMP levels with 0.3 mM 8-bromo-cAMP affected neither the depolarization-induced release of 14C-ACh or 3H-DA from accumbens slices nor the inhibitory effects of opioid receptor activation thereon. The results indicate that the mechanism by which functional mu and delta receptors presynaptically inhibit the depolarization-induced 14C-ACh release from nucleus accumbens slices is likely to involve an increase of potassium channel conductance. In contrast, activation of kappa-opioid receptors, which inhibits depolarization-evoked 3H-DA release, apparently does not result in a hyperpolarization of (dopaminergic) nerve terminals. In none of these inhibitory effects presynaptic adenylate cyclase appears to be involved.


Antiarrhythmic and electrophysiological effects of SD-3212, a novel Na+ and Ca++ channel blocker, in anaesthetized dogs with myocardial infarction in comparison with its stereoisomer (SD-3211) and bepridil. Antiarrhythmic and electrophysiological effects of SD-3212, a novel antiarrhythmic agent, which has both Na+ channel and Ca++ channel blocking activities, were compared with those of its (+)-stereoisomer, SD-3211, which has only a Ca++ channel blocking activity, and bepridil, a known Ca++ channel blocker with additional Na+ channel blocking activity, using the two-stage coronary ligation induced arrhythmia (24 h after the ligation of the left anterior descending coronary artery) and 7 day-old myocardial infarcted hearts in anaesthetized dogs. SD-3212 showed a dose-dependent antiarrhythmic effect on the two-stage coronary ligation induced arrhythmia. SD-3212 at a dose of 3 mg/kg reduced the arrhythmic ratio, i.e. ectopic beats per min divided by the sum of ectopic beats and sinus beats per min, significantly from 1 up to 12 min after the administration. Neither bepridil (1-6 mg/kg) nor SD-3211 (1 mg/kg) had an antiarrhythmic effect. SD-3212 (0.3-3 mg/kg) prolonged both the conduction time in the normal myocardium and the delayed potential in the infarcted myocardium in the 7 day-old myocardial infarcted hearts in anaesthetized dogs in a dose-dependent manner. This effect of SD-3212 was shown at coupling intervals of 150-1000 ms increasing with decreasing interval. In this respect, SD-3212 is similar to drugs which show fast recovery of Vmax from use-dependent block such as lidocaine. Bepidril (1-6 mg/kg) also prolonged these parameters in a dose-dependent manner, however, the prolongation induced by bedripil was limited to shorter coupling intervals as compared with that induced by SD-3212. SD-3212 (0.1-1 mg/kg) did not show this prolonging effect.(ABSTRACT TRUNCATED AT 250 WORDS)


Metitepine distinguishes two receptors mediating inhibition of [3H]-5-hydroxytryptamine release in guinea pig hippocampus. Inhibition of [3H]-5-hydroxytryptamine ([3H]-5-HT) release from guinea pig brain slices via activation of the terminal 5-HT autoreceptor has previously been characterised as a model of 5-HT1D receptor activation, based on the rank potencies of a range of agonists, and the potent antagonism of the inhibitory effects of 5-HT by metitepine. The present study uses this model, in slices of the guinea pig hippocampus, to examine the antagonist potency of metitepine against the 5-HT receptor agonists sumatriptan, 5-carboxamidotryptamine (5-CT) and 5-HT. Addition of metitepine to the perfusion buffer (30, 300 and 1000 nmol/l) significantly shifted the concentration-response curve to 5-HT, producing a Schild slope of 1.1, and a pA2 value of 7.6. However, the ability of metitepine to antagonise the effects of sumatriptan or 5-CT in this model was less marked. A clear-cut shift in the concentration-response curve to sumatriptan was only achieved at 1000 nmol/l metitepine (apparent pA2 = 6.7), and this was similar to the ability of metitepine to attenuate the effects of 5-CT (apparent pA2 7.0 at 300 nmol/l and 6.7 at 1000 nmol/l). These findings suggest heterogeneity in the receptor mediating inhibition of [3H]-5-HT release in guinea pig hippocampus.


Inhibition of N-methyl-D-aspartate- and kainate-evoked noradrenaline release in human cerebral cortex slices by ethanol. The effect of ethanol on the release of noradrenaline evoked by various stimuli was investigated in human cerebral cortical slices from patients undergoing neurosurgery. The slices were preincubated with [3H]noradrenaline and then superfused. Tritium overflow was stimulated by exposure to N-methyl-D-aspartate (NMDA; in slices superfused without Mg2+), kainic acid, veratridine or by increasing the K+ concentration. The NMDA-evoked tritium overflow was concentration-dependently inhibited by ethanol; an inhibition by 37% occurred at 48 mmol/l ethanol. This ethanol concentration was not yet effective when kainic acid was used for stimulation, but ethanol 150 mmol/l strongly inhibited the tritium overflow evoked by kainic acid as well. The tritium overflow evoked by veratridine or high K+ was not affected by ethanol in the concentration range investigated. These findings are compatible with the suggestion that the NMDA receptor and, with less susceptibility, the kainate receptor are sites of action underlying the effect of ethanol in the human brain.


[Intra-lesion administration of beta-interferon in the treatment of CIN associated with HPV infection]. Thirty-two women with histologically confirmed cervical intraepithelial neoplasia (CIN) associated with human papillomavirus (HPV) infection were treated with intralesional beta-interferon. At 12 months from the end of the treatment, 60% of the patients showed complete regression, histologically assessed, of CIN. Considering separately the different CIN grades, the regression for CIN I was 71%, 64% for CIN II and 45% for CIN III. Side-effects were rather frequent (84%) but they did not require discontinuation of the treatment. On the basis of these data the Authors believe that intralesional beta-interferon, in selected cases, can play a role, as a conservative modality, among the different techniques of CIN therapy.


Positron emission tomographic studies of central cholinergic nerve terminals. The aim of this study was to develop a quantitative method for the study of cholinergic nerve terminals in vivo. An 18F-labeled analogue of vesamicol ([18F]FMV) that binds with high affinity to synaptic vesicles from Torpedo electric organ was synthesized and evaluated in vivo in rats and monkeys by positron emission tomography (PET). In rats, the tracer was rapidly cleared from the blood and highly extracted into the brain, where it was specifically and irreversibly bound. In monkeys, a specific binding of the tracer was observed in brain regions known to contain cholinergic nerve terminals. Preinjection of non-labeled vesamicol prevented the cerebral binding of [18F]FMV to a high affinity site in both species. Our results are a major step towards quantitative human in vivo studies of presynaptic cholinergic functions.


Sensitive procedures for measuring chloride fluxes mediated by the purified glycine receptor incorporated into phospholipid vesicles. Two novel methods have been developed to directly measure chloride influx into purified glycine receptor-containing phospholipid vesicles. Using a method based on the fluorescence quenching of a chloride-sensitive probe entrapped into the vesicles, a chloride influx was detected which could be enhanced by glycine and completely abolished by the antagonist strychnine. In addition, by tracing the 36Cl- influx into the proteoliposomes, a stimulatory effect of both glycine and beta-alanine could be seen, which can be inhibited by strychnine and other glycine antagonists. These data, together with a previous report demonstrating ligand-mediated iodide fluxes in the same preparation (Biochemistry, 28 (1989) 6405-6409), clearly demonstrate the utility of the reconstituted receptor preparation to investigate some ion channel and pharmacological properties of the glycine receptor.


Nerve growth factor-induced loss of cell-associated nerve growth factor receptor in human melanoma A875 cells. Human A875 melanoma cells are known to express the low-affinity nerve growth factor receptor p75NGFR in a monomeric and a covalently linked probably dimeric form. Kinetic analysis of the association of nerve growth factor (NGF) with its receptor revealed a rapid loss of binding sites at high ligand concentrations. Using cross-linking and immunoprecipitation with an anti-p75NGFR antibody, this was found to be due to a decrease of the high molecular weight form of the receptor. Mechanisms for such a ligand-induced receptor loss are discussed.


Localization of (-)-[125I]cyanopindolol binding in guinea-pig heart: characteristics of non-beta-adrenoceptor related binding in cardiac pacemaker and conducting regions. Receptor autoradiography was used in guinea-pig heart to locate binding sites for the beta-adrenoceptor ligand (-)[125I]cyanopindolol (CYP) resistant to blockade by the beta-adrenoceptor antagonist (-)-propranolol (1 microM). Highly localized binding was observed to regions closely associated with the sinoatrial node, atrioventricular node and bundle of His but was not observed on myocardial, pacemaker, conducting cells or adipose tissue. Free [125I] also bound to identical sites. Binding was enhanced in the presence of ascorbic acid but was completely inhibited by (-)-isoprenaline (100 microM), serotonin (5-HT) (10 microM) and phentolamine (10 microM).


GM1 ganglioside treatment promotes recovery of electrically-stimulated [3H]dopamine release in striatal slices from rats lesioned with kainic acid. The electrically-evoked release of [3H]dopamine ([3H]DA) from rat striatal slices was studied after a monolateral intrastriatal injection of kainic acid (KA). The release in the KA-lesioned striatum measured 4 days after the lesion was largely reduced (by 80%) with respect to the contralateral striatum. Administration of GM1 ganglioside (GM1) beginning on the day of the lesion resulted in restoration of the catecholamine release. Significant recovery was observed when GM1 was administered i.p. daily at the dose of 3 mg/kg for 6 days. The ganglioside given for 6 days at 30 mg/kg restored to near normal the electrically-evoked [3H]DA release. Similar recovery from the KA-induced injury occurred spontaneously but required 50 days.


Clinical and pathological study of two patients with progressive supranuclear palsy and Alzheimer's changes. Antigenic determinants that distinguish cortical and subcortical neurofibrillary tangles. Two cases with classical clinical manifestations of progressive supranuclear palsy (PSP) showed severe progressive dementia as an additional clinical feature. Neuropathological study demonstrated typical features of PSP in the brainstem. Additionally, histological criteria of Alzheimer's disease (AD) were observed. A topographic and immunohistological study (with neurofilament subunit and Tau and Ubiquitin antibodies) of the distribution of neurofibrillary tangles (NFTs) was performed in order to compare the characteristics of NFTs from cortex and brainstem. NFTs from cortex were positive with all antibodies used and were predominantly distributed in cortical layers III and V and affected medium size neurons. Brainstem NFTs were positive only for neurofilament subunits and Tau. Cortical and brainstem NFTs showed immunohistological differences. Cortical NFTs in our two cases had a similar distribution as in control AD cases. On the basis of our observations we believe (1) that cortical tangles in our PSP cases are related to Alzheimer's disease and (2) that the cortical NFTs of PSP and AD are morphologically and immunohistologically distinct. Mechanisms concerned with the production of cortical and brainstem NFTs in PSP and AD are discussed.


Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus. In situ hybridization procedures indicate that estrogen selectively increases neurotensin and neuromedin (NT/N) mRNA levels in the rostral preoptic area of the rat hypothalamus (RPH). Using the co-localization procedures of Axelson and Van Leeuwen, J. Neuroendocrinol., 2 (1990) 209-216, the present study examined whether NT cells in the RPH contained estrogen receptors (ER). Vibratome sections of brains from adult ovariectomized, colchicine-treated rats were first incubated with estrogen receptor antibody and stained with diaminobenzidine (DAB)-Ni+ producing a blue-black nucleus. Subsequently, NT antisera were used to provide a brown reaction product with DAB as chromogen. Approximately 25% of the NT cells in the RPH contained ER. These data support the hypothesis that NT cells in the RPH that play a role in luteinizing hormone release from the pituitary are, in part, influenced directly by estrogen feedback via nuclear ER and may act as interneurons controlling luteinizing hormone releasing hormone turnover.


Ubiquitin expression in globose tangles in the locus coeruleus in brains of patients with Alzheimer's and Parkinson's diseases. The locus coeruleus belongs to brain areas exhibiting remarkable neuronal loss already during physiological aging and very early neurofibrillary tangles. We performed a semiquantitative, immunocytochemical study focused on the expression of ubiquitin in neurofibrillary tangles in the locus coeruleus as compared with the central superior nucleus. The locus coeruleus exhibited later ubiquitin expression in the neurofibrillary tangles, a lower percentage of ubiquitinated tangles and more frequently granular cytoplasmic staining with Tau-1 than the central superior nucleus. These results suggesting different, probably delayed, processing of Tau protein in the locus coeruleus might contribute additionally to cell injury in this area.


Immunohistochemical study of A2B5-positive ganglioside in postmortem human brain tissue of Alzheimer disease, amyotrophic lateral sclerosis, progressive supranuclear palsy and control cases. Localization of gangliosides positively stained by the monoclonal antibody A2B5 was investigated in postmortem brain tissue of Alzheimer disease, amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP) and control cases. In control cases, A2B5-staining was granular, appearing in selective neuronal populations. In the neocortex, the A2B5-positive neurons were distributed mainly in deep cortical layers. In the cerebellum, A2B5-positive structures were detected in processes extending from the Purkinje cell layer into the molecular layer. In Alzheimer cases, many neurofibrillary tangles, neuropil threads and dystrophic neurites were strongly A2B5-positive. In addition, aggregations of A2B5-positive granules were detected in some neurons lacking neurofibrillary tangles. Alterations of A2B5-positive gangliosides were also detected in ALS and PSP cases. In ALS cases, A2B5-positive granules were aggregated in Betz cells of the precentral gyrus. In PSP cases, globose-type neurofibrillary tangles were also strongly A2B5-positive. The results indicate that A2B5-positive gangliosides are widely but selectively distributed in human brain and may be involved in several neuropathological processes.


Amyloid beta-protein precursor deposition in rat hippocampus lesioned by ibotenic acid injection. Ibotenic acid was injected into 3 parts of the lateral rat hippocampus. The animals were sacrificed 100 days after treatment, and studied immunohistochemically. The lesioned side of the hippocampus was highly atrophic with extensive neuronal loss and gliosis. Although silver staining revealed no particular structures such as neurofibrillary tangles or senile plaques, globular and granular depositions of amyloid beta-protein precursor (APP) immunoreactivity was observed by immunostaining in the lesion with the monoclonal antibody (clone 22C11). Increased immunoreactivities of glial fibrillary acidic protein (GFAP) and ubiquitin were found in the lesioned area, while the immunoreactivities of microtubule-associated protein 2 (MAP2) and 200 kDa neurofilament subunit protein (NF-H) were diminished. The results indicate that APP deposition is formed in the lesioned area where neuronal degeneration is produced by ibotenic acid in rat hippocampus.


Congenital infections and the nervous system. Despite vaccines, new antimicrobials, and improved hygienic practices, congenital infections remain an important cause of death and long-term neurologic morbidity among infants world-wide. Important agents include Toxoplasma gondii, cytomegalovirus, Treponema pallidum, herpes simplex virus types 1 and 2, and rubella virus. In addition, several other agents, such as the varicella zoster virus, human parvovirus B19, and Borrelia burgdorferi, can potentially infect the fetus and cause adverse fetal outcomes. This article provides an overview of these infectious disorders and outlines current strategies for acute treatment and long-term management.


A comparison of herpes simplex virus specific antibodies found in human milk and serum. It is not known if milk antibody protects infants from herpes simplex virus (HSV) infection. As a first step to test this hypothesis, anti-HSV antibodies were studied in human milk. Paired serum and milk samples were analyzed for anti-HSV antibodies by ELISA, Western blot analysis (WBA), neutralization (NT) plaque assay, and antibody-dependent cellular cytotoxicity (ADCC) assay. Nineteen of the 20 serum samples showed anti-HSV activity by ELISA and ADCC, and 18 showed activity by WBA and NT. We found a significant association between the immunoassays for detection of anti-HSV antibodies in sera. Fewer of the human milk samples showed anti-HSV activity; only one milk sample was positive by ELISA and one by NT assay, four by ADCC and 12 by WBA. The milk sample from the seronegative donor was also negative. We found a poor association of antibody titers in human milk and serum antibody titers using ELISA, NT, and ADCC assays. There was a significant (p = 0.022) association between serum and milk results using WBA. Among the four assays, WBA was the most sensitive for antibody detection. It will be used in an on-going prospective study to determine the role of anti-HSV antibody in the protection against HSV infections in infants.


Myocardial cholinergic signaling changes with age. We examined the linkage of cholinergic receptors to the phosphoinositide signaling pathway to elucidate one facet of the autonomic response mechanism in fetal and adult sheep. Cholinergic stimulation with carbachol increases the production of 3H-inositol mono-, bis-, and trisphosphates in a time- and concentration-dependent manner in both fetal and adult myocardium. However, the maximal stimulation of inositol polyphosphates above basal activity was much greater in fetal (120 +/- 11%) than in adult (20 +/- 7%) myocardium (mean +/- SEM). Saturation binding analysis of myocardial muscarinic receptors using 3H-N-methylscopolamine revealed significantly higher receptor concentration in fetal (240 +/- 25 fmol/mg protein) than in adult (78 +/- 15 fmol/mg protein) myocardium (mean +/- SEM). Binding competition studies revealed a pattern of selectivity-atropine less than 4-diphenylacetoxy-N-methylpiperidine methiodide less than pirenzepine less than or equal to (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride less than or equal to 11-2[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one 116-compatible with the presence of muscarinic receptor (MR)2, MR3, and/or MR5 subtypes. Receptor subtype determination by Northern blot analysis revealed mRNA specific for the MR2 subtype in both fetal and adult myocardium, although expression was greater in fetal heart. We conclude that decreases in MR2 subtype protein and mRNA levels parallel the age-related decrease in carbachol-stimulated PLC activity. Our studies demonstrate differences between fetal and adult myocardium in the concentration of muscarinic cholinergic receptors and their linkage to a putative calcium mobilizing signaling pathway and suggest that this pathway may play a different role in the fetus than in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)


Corticotropin releasing hormone stimulation test and nocturnal cortisol levels in normal children. This study examined hypothalamic-pituitary-adrenal axis functioning in a group (n = 25) of very carefully screened normal children with considerable attention to issues of adaptation and procedural stress. The subjects (mean age 10.3 +/- 1.6 y) were selected as "supernormal" controls as a part of a large psychobiologic study of childhood depression. After careful acclimatization over 24 h, the subjects underwent all-night sampling of plasma cortisol every 20 min, then the following evening had a corticotropin releasing hormone (CRH) stimulation test (using human CRH). Human CRH resulted in a rapid stimulation of adrenocorticotropin and cortisol. Adrenocorticotropin levels increased from 6.8 +/- 3.5 (+/- SD) pmol/L (30.7 +/- 16.1 pg/dL) to a peak of 11.6 +/- 5.5 pmol/L (52.9 +/- 24.8 pg/mL) at 15 min with return to baseline levels by 60 min. Cortisol levels increased from 131.4 +/- 59.7 nmol/L (4.8 +/- 2.2 micrograms/dL) to a peak of 427.0 +/- 113.5 nmol/L (15.5 +/- 4.1 micrograms/dL) at 30 min with return to baseline by 120 min. The cortisol peak was significantly greater (p less than 0.05) in boys [474.6 +/- 129.7 nmol/L (17.2 +/- 4.7 micrograms/dL)] than in girls [366.9 +/- 52.4 nmol/L (13.3 +/- 1.9 micrograms/dL, p less than 0.05)]. Age, body mass index, and pubertal status were not significantly related to hypothalmic-pituitary-adrenal axis measures. Nocturnal cortisol reached a nadir at 160 +/- 60 min after sleep onset (0102 h) and a peak 480 +/- 60 min after sleep onset (0612 h). Nocturnal cortisol levels were significantly (positively) correlated with human CRH-stimulated cortisol (r = 0.56, p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)


[Color-coded duplex sonography of liver tumors. Does the blood supply permit a conclusion on staging?]. Real-time ultrasonography is a sensitive screening method in patients with suspected liver tumors. However, sonomorphology does not differentiate between benign and malignant tumors. In a prospective study we examined patients with liver tumors with color-coded duplex ultrasonography to find out whether the perfusion status of the liver tumor permits a differential diagnosis. A total of 108 patients with liver tumors were included, and the results of color-coded duplex sonography were compared with histology, MRI and CT. No correlation was seen between the final diagnosis and the perfusion pattern. The vascular status visualized by color-coded duplex ultrasonography does not permit differentiation between benign and malignant liver tumors.


Dynamic CT of the liver.

Dynamic CT of the liver. The most commonly used CT technique for contrast enhancement CE of the liver are dynamic CT scanning after either IV, direct hepatic arterial or indirect portal contrast injection. The need for these different approaches is because compromises have to be made which take into account the scanner performance, the pharmacodynamics of the available urographic contrast agent and the complex vascularisation of the liver and the tumors. Spiral scanning is a novel technique which allows to scan the entire liver in a single breathhold period of about 10 to 20 seconds with the a posteriori calculation of perfectly contiguous or even overlapping slices. These two new specifications offer entirely new perspectives in terms of liver enhancement. It becomes theoretically possible to scan the entire liver twice, during both the early arterial phase and the subsequent venous phase after one single short IV bolus injection. Hence the total amount of contrast media to be administered can be reduced while the diagnostic potential of the contrast agent is significantly increased.


CT during arterial portography: diagnostic pitfalls. Computed tomography (CT) during arterial portography (CTAP) is an important technique for evaluating the liver before hepatic tumor resection. With this technique, most tumors are of low attenuation compared with that of enhancing parenchyma. At times, low-attenuation lesions are encountered that represent perfusion abnormalities rather than tumor deposits. These perfusion abnormalities can be categorized as (a) those resulting from improper technique; (b) those extending from hilum to capsule (straight-line sign), with or without an obstructing mass; (c) perihilar and periligamentous abnormalities; (d) subcapsular defects (linear or wedge shaped); and (e) those seen with cirrhosis or regenerating nodules. Adjuvant use of delayed CT, magnetic resonance imaging, and intraoperative ultrasound aids in characterization of these nontumorous defects, thereby improving specificity. The authors conclude that when potential candidates are evaluated for hepatic tumor resection, knowledge of the existence of the various diagnostic pitfalls of CTAP and their imaging characteristics is imperative to avoid inadvertent false results.


Treatment options in high-grade brain tumors: brain brachytherapy. The extremely poor prognosis of high-grade brain tumors (glioblastoma multiforme and anaplastic astrocytomas) has been well documented in the literature. Almost 90% of patients die within 18 months after therapy, most commonly because of local persistence of the tumor, which may be controlled if a sufficient amount of irradiation can be delivered. Currently, postoperative radiation therapy offers the best median survival rate. However, the response to external-beam radiation therapy has reached a plateau because of the intolerance of healthy brain tissue to excessive irradiation. To treat these tumors, brachytherapy (interstitial implantation of radioactive sources) can be used with debulking surgery. This therapy is becoming an effective alternative to conventional external-beam radiation therapy, since it allows a higher dose to be delivered to the tumor bed without damaging the surrounding healthy brain tissue. With continual refinements of the technique, brachytherapy, performed by a skilled brachytherapy team, offers an opportunity to improve patient survival.


Modulation of kappa-mediated antitussive activity in rats by a delta-agonist. When co-administered intracisternally, the selective delta-opioid agonist [D-Pen2,5]enkephalin (DPDPE), which had no significant effect on the cough reflex, consistently and significantly decreased the antitussive potencies of kappa-receptor agonists, U-50,488H and U-62,066E. The decrease in the antitussive effects of these kappa-receptor agonists caused by DPDPE were prevented by selective delta receptor antagonist, naltrindole. These results suggest that delta receptors may play an inhibitory role in antitussive processes that are mediated by the kappa-receptors.


The adenylate cyclase system and prostaglandin production in human decidua parietalis. We studied the beta-adrenergic system in the human decidua and its effect on prostaglandin E2 and F2a release from dispersed decidual cells in culture. Beta-adrenergic receptors in decidual membranes were partially characterized using (+)[125I]HYP. Specific binding was demonstrated with maximum binding capacity (Bmax) of 70 +/- 10.6 fmol/mg and an affinity (KD) of 20.85 +/- 1.86 pmol. cAMP dependent phosphoproteins in decidual cytosol were also identified. Two phosphoproteins of M(r) 42,000 and 22,000 were seen in all preparations. Three others (M(r) 39,000, 23,000 and 21,000) were identified in only some of the preparations. Phosphoproteins of similar M(r) to those seen in cytosol prepared from decidual homogenates were also identified in cytosol of cultured decidual cells. Phosphorylation of the 42,000 M(r) and 22,000 M(r) proteins was maximal (3.04 +/- 0.35-fold and 5.7 +/- 0.68-fold) with 10(-6) M cAMP. Cultured decidual cells produced prostaglandin E2 and F2a which increased in a dose-dependent manner in response to dbcAMP, forskolin or isoproterenol. The decidua contains an intact beta-adrenergic system that, when activated, is capable of phosphorylating specific proteins and modulating prostaglandin release.


Effect of serum on cell membrane Na-K transport of vascular smooth muscle in culture--a comparative study between normotensive and hypertensive rats. In order to elucidate the effect of serum and its differential characteristics in primary hypertension, we investigated the influence of serum from spontaneously hypertensive rats (SHR) on the cell membrane Na-K pump, Na-H antiport and passive K permeability of cultured vascular smooth muscle cells (VSMC) from SHR and Wistar-Kyoto rats (WKY). In the absence of serum, the Na-K pump activity, described as ouabain-sensitive 86Rb uptake (OS), was greater in the VSMC of the SHR than that of the WKY. Addition of serum to the quiescent VSMC stimulated the OS, of which activation was significantly greater with the SHR serum than with the WKY serum. Determination of intracellular Na concentrations in the presence of 1 mM ouabain showed that Na uptake in the absence of serum of the SHR VSMC was greater than that of the WKY VSMC. Subjecting the VSMC to rat serum stimulated Na uptake, which effect was more profound with SHR serum than with WKY serum. A greater stimulation of 5-(N,N-hexamethylene) amiloride(HMA)-sensitive Na uptake by SHR serum accounted for this difference. When the Na-K pump was active, intracellular Na concentrations were unchanged in the presence of serum. Furthermore, serum from either of the two strains produced a substantial increase in the washout rate constant of 86Rb washout (Ke), which effect was also larger in SHR serum than in WKY serum. In the absence of serum, basal Ke of the SHR VSMC was greater than that of the WKY VSMC. It is concluded that serum from SHR produced a more pronounced activation of the Na-K pump, Na-H antiport and passive K permeability in cultured VSMC. The study suggests that in addition to an innate augmented activity of Na-K transporters in the VSMC of SHR, there are humoral factors in SHR serum which elicit the stimulation of the Na-H antiport, leading to either the activation of the Na-K pump or secondary stimulation of passive K permeability across the cell membrane.


Verapamil attenuates postischemic oxidative injury in the rat liver. We assessed the effects of the calcium channel blocker verapamil on postischemic oxidative injury in the rat liver. In the untreated rats, the values of tissue lipid peroxidation products (thiobarbituric acid-reactive substances) remained unchanged during 90 min of warm ischemia. However, the values increased significantly after the next 60 min of reperfusion compared with those in the sham-operated rats (P less than 0.01). Intravenous infusion of verapamil (5 micrograms.kg-1.min-1) significantly reduced the extent of lipid peroxidation during reperfusion compared with that in the untreated rats (P less than 0.02). The percentages of tissue water content and the serum lactate dehydrogenase activities after 60 min of reperfusion were significantly lower in the treated rats than in the untreated rats (P less than 0.02 and P less than 0.01, respectively). We also investigated the influence of verapamil on superoxide-generating activity determined by the superoxide-dependent cytochrome c reduction of peritoneal polymorphonuclear leukocytes (PMNs) harvested from normal, non-ischemic, and non-treated rats in vitro. This demonstrated that there was no apparent effect with the highest verapamil concentration level (8 microM) observed in the rat plasma during our experiment. These findings suggest that verapamil might reduce the postischemic oxidative injury in the rat liver by mechanisms perhaps not related to the suppression of rat PMNs superoxide-generating activity.


Isolation of eastern equine encephalitis virus from Aedes albopictus in Florida. Fourteen strains of eastern equine encephalitis (EEE) virus were isolated from Aedes albopictus mosquitoes collected in Polk County, Florida. These are the first isolations of an arbovirus of proven public health and veterinary importance from naturally infected Ae. albopictus in the United States since established populations of this introduced mosquito were first discovered in 1985. The widespread distribution of Ae. albopictus in Florida and in other areas of the United States where EEE is endemic raises concern that this species may become an epizootic and epidemic vector of EEE virus.


Anti-human immunodeficiency virus therapeutics: now and the future. Knowledge of the HIV life cycle helps to plan rational modalities directed against the virus. The complex nature of the life cycle of HIV presents multiple unique targets. HIV binding to the cellular receptor CD4, viral enzymatic targets, HIV activation, viral protein synthesis, and protein packaging are examples of the types of targets available to inhibit the life cycle of HIV. Patients, community groups, regulatory agencies, national research groups, and the private pharmaceutical companies have joined forces to work to a common goal, effective HIV therapies. RT inhibitors have provided the best therapeutic options, but other drugs are being developed at a rapid pace. New drugs are entering clinical investigation and will be more widely available at early signs of clinical and laboratory effectiveness.


The development of cellular polarity in transport epithelia. From the above discussion, it can be seen that the establishment of epithelial polarity plays a critical role in mammalian development. Functional and structural transitions in the vectorial processes of epithelial tissues in the developing embryo accompany critical events during development. Pathophysiologic states may arise due to either genetic or acquired abnormalities of polarity. Immaturity of vectorial transport functions frequently accompany the onset of premature birth and result in abnormal function of transport epithelia. Further definition of the molecular mechanisms that lead to the establishment and maintenance of epithelial polarity, as well as the developmental sequence of vectorial transport functions in developing epithelia, will lead to better understanding and treatment of fundamental disease states in the fetus and newborn.


Long term analysis of factors influencing the outcome in carcinoma of the breast smaller than one centimeter. We evaluated established risk factors (tumor size, lymph node status, menopausal status, estrogen receptor status, tumor histology and grading according to Bloom and Richardson, including subfactor analysis), as well as the influence of local procedures, in 138 patients with primary carcinoma of the breast smaller than 1 centimeter. The patients were operated upon during 1969 to 1989 at the Department of Surgery, Hanusch Medical Center, Vienna. Twenty-two patients had a recurrence after a median observation time of 15 years. Seven patients died of the primary disease by the control date (31 May 1990). Grading (p = 0.01, 0.0044) as well as nuclear polymorphism (p = 0.003, 0.00001) and mitosis rate (p = 0.02, 0.01) proved to be significant parameters for recurrence free survival and overall survival. Local procedures (modified radical mastectomy, breast conserving operation with or without postoperative radiotherapy) revealed borderline significance with local recurrence free survival (p = 0.08). All other parameters were without any statistical significance (Mantel-Cox log rank test). Our data confirm the superior prognostic relevance of histologic grading and nuclear polymorphism in patients with carcinoma of the breast smaller than 1 centimeter. High grade nuclear polymorphism as a subfactor in the grading classification according to Bloom and Richardson appears to be a highly valid risk factor for this entity.


Adenosine-mediated synaptic inhibition: partial blockade by barium does not prevent anti-epileptiform activity. Adenosine-induced inhibition of evoked postsynaptic potentials (PSPs) and epileptiform burst firing in the CA1 subfield of rat hippocampal slices was studied with intracellular recordings in vitro. Adenosine (50 microM) caused a membrane hyperpolarization which was abolished during superfusion with 2 mM Ba2+. The adenosine-induced inhibition of the PSPs was still evident, although the magnitude of the effect was significantly reduced. Adenosine also reduced Ba(2+)-induced burst firing, but less effectively than it did bursts evoked by TEA (5 mM). The results suggest that adenosine inhibits synaptic transmission and epileptiform activity by at least 2 mechanisms: a postsynaptic barium-sensitive increase in gK and a presynaptic effect independent of this adenosine-evoked outward potassium conductance.


Acute stress impairs (or induces) synaptic long-term potentiation (LTP) but does not affect paired-pulse facilitation in the stratum radiatum of rat hippocampus. Rats were exposed to restraint coupled with 60, 1-sec, 1-mA, 60-Hz tail shocks. One hippocampus was immediately dissected for in vitro measurement of paired-pulse facilitation and LTP of the excitatory postsynaptic potential (EPSP) recording from the stratum radiatum of field CA1. There was no change in paired-pulse facilitation, suggesting that acute exposure to the stressor does not result in a decrease in presynaptic neurotransmitter release. There was, however, a significant decrease in the percent LTP produced by theta burst stimulation relative to naive controls. These results are consistent with the hypothesis that the stress-induced impairment of LTP is a result of changes in the postsynaptic glutamate receptors, specifically the AMPA type.


Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation. The effect of prostaglandin E1 (PGE1) on platelets is mediated through the PGE1 receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE1 and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 microns. The time course of the extension length (Dp, in microns) of the platelets in response to aspiration with a negative pressure (delta P) of 5 cm H2 O (delta P x Rp = 0.15 dynes/cm) was analyzed. PGE1 treatment (0.1 microM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE1-treated)/Dp/Rp (control), was significantly reduced to 0.90 +/- 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE1-treated platelets had a higher [cAMP]i than controls. Platelets treated with PGE1 or dbcAMP showed a reduced [Ca2+]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE1 and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]i and low [Ca2+]i after PGE1 treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.


Study of anticoagulant mechanism of low molecular weight heparin. The binding ability of low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) to factor Xa (F.Xa), thrombin and AT III was investigated using FR-860- and UF-heparin-Sepharoses. FR-860 could not bind directly to F.Xa. FR-860 bound to thrombin and AT III with stronger affinity to AT III than to thrombin. On the other hand, UF-heparin bound to F.Xa, thrombin and AT III with the strongest affinity to AT III followed by thrombin and F.Xa. AT III mediated the binding between F.Xa and FR-860 and accelerated the reaction between F.Xa and UF-heparin. On the other hand, AT III did not affect the binding between thrombin and FR-860 or UF-heparin. Diisopropyl fluorophosphate-treated thrombin inhibited the binding between AT III and FR-860, but not that between AT III and UF-heparin. These results suggest that the anti-F.Xa activity of FR-860 is mediated by AT III. Furthermore, the difference of antithrombin activity between FR-860 and UF-heparin depends on the capability to form ternary complex of FR-860 or UF-heparin, AT III and thrombin.


The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia. The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.


A pathological and immunohistological case report of fatal infectious mononucleosis, Epstein-Barr virus infection, demonstrated by in situ and Southern blot hybridization. We present an autopsy case of 20-month-old boy who had a fulminant course of infectious mononucleosis, with severe hepatic failure. Autopsy revealed marked infiltration of immunoblasts in the lymph nodes, liver, spleen, thymus and kidneys. We identified a large number of Epstein-Barr virus (EBV) genomes in the immunoblasts of the lymph nodes, liver and spleen by in situ hybridization. EBV genomes were also detected in the liver and spleen by Southern blot hybridization. Histology of the liver revealed diffuse feathery degeneration of the hepatocytes. However, EBV genomes were not detected in the hepatocytes by in situ hybridization and monoclonal antibody studies. Immunostaining of the autopsy liver specimen revealed a large number of suppressor/cytotoxic T cells (Leu2a positive) in the portal areas and of natural killer (NK) cells (Leu7 positive) in the portal areas and sinusoids of the liver. We therefore suggest that the hepatocellular damage was not caused by the viral replication in the hepatocytes but was mainly caused by the abnormal killer cell activity of the suppressor/cytotoxic T cells and NK cells.


Pericapillary rosettes in the human spinal cord. We have found large eosinophilic bodies in the pericapillary regions of the gray and white matter in the human spinal cord. These are entirely different from the previously reported pericapillary inclusion bodies (PIB). We have designated them pericapillary rosettes (PR), since they consist of clusters of round or ovoid structures of various sizes which form rosettes around the transversely sectioned capillaries. Positive staining with silver impregnation and anti-neurofilament antibody, the presence of the myelin around the bodies and the accumulation of neurofilaments within them indicate that axonal swelling is associated with the formation of PR. These bodies are not observed in individuals aged less than 30 years, and afterwards they increase in proportion to age. Over the age of 80, they are almost always found in the spinal cord, particularly in the gray matter of both the anterior and posterior horns at the thoracic and lumbar levels. Thus, the development of PR is closely related to the aging process.


Morphological findings on peripheral nerve biopsies in 15 patients with human immunodeficiency virus infection. A peripheral nerve biopsy was performed in 15 patients with human immunodeficiency virus (HIV) infection and polyneuropathy. Two cases [1 asymptomatic, 1 AIDS-related complex (ARC)] presented with chronic inflammatory demyelinating polyneuropathy; there was 1 case (asymptomatic) of mononeuropathy multiplex and 12 cases (1 asymptomatic, 1 ARC, 10 AIDS) with distal symmetrical polyneuropathy. Epi- or endoneurial microvasculitis was observed in 6 cases. Electron microscopy showed that nerve fiber lesions were mainly axonal. Severe segmental demyelination was also present in both cases of chronic inflammatory demyelinating polyneuropathy, with characteristic features of active demyelination in one. Numerous plasmacytoid cells were found in the endoneurium in 4 patients. Tubuloreticular inclusions were present in endothelial cells in the 10 cases with AIDS but absent in the other patients. Direct immunopathological examination with anti-immunoglobulin sera was negative in all cases. HIV was evidenced by in situ hybridization in 2 AIDS patients; no Epstein-Barr virus or cytomegalovirus was detected.


Cytologic diagnosis of human papillomavirus. Influence of age and pregnancy stage. In a review of cytologic findings in 446,038 premenopausal women whose smears were examined during a five-year period ending in 1990, it was established that cytomorphologic evidence of human papillomavirus infection was significantly more common in the younger groups: those under 30 years of age had about twice the rate of the older groups. Similarly, in a group of 29,153 pregnant women it was found that cytologic signs of human papillomavirus were reported twice as often during the second half of pregnancy as in the first.


The use of nerve growth factor as a reverse transforming agent for the treatment of neurogenic tumors: in vivo results. The rationale behind the evaluation of natural differentiating agents, such as nerve growth factor (NGF), for reverse transforming potential is based on the theory that such compounds may represent a nontoxic means of controlling tumor growth. Previous in vitro experiments have shown that NGF is capable of retarding growth and of inducing persistent differentiation of neurogenic tumor cell lines. In vivo, NGF is capable of causing a persistent reduction in the number of ethylnitrosourea-induced neurinomas and of increasing survival time following intracerebral implantation of F98 anaplastic glioma cells. In this study, anaplastic glioma and neurinoma implants were treated with NGF to evaluate the reverse transforming potential of NGF in vivo. Results indicate that NGF is capable of causing a significant decrease in the growth rate of subcutaneous T9 (anaplastic glioma) and clone 16 (anaplastic neurinoma) implants. Significantly, NGF treatment was accompanied by adverse effects that were minimal and transient. Continued tumor growth (although greatly retarded) following NGF treatment is an aspect that requires further investigation. However, the results of this study suggest that NGF may prove useful, alone or in combination with other types of therapy, for the treatment of tumors of neurogenic origin.


Intracisternal inclusions in Schwann cells of the sural nerve. Biopsy of the sural nerve in a 74-year-old man with chronic neuropathy demonstrated Schwann cells that possessed multiple cisterns of rough endoplasmic reticulum filled with 7- to 8-nm filaments and perinuclear cisterns that were markedly distended with fine granular substances and electron-dense globules. To our knowledge, this is the third case exhibiting filamentous inclusions in Schwann cells. Unlike the previous cases, however, this case showed inclusions in the distended perinuclear cisterns and axonal degeneration. The significance of these alterations remains to be elucidated.


Fine needle aspiration cytology of primitive neuroectodermal tumors. A report of these cases. Primitive neuroectodermal tumor (PNET) is a small round cell malignancy arising in soft tissue and bone, predominantly in older children and adolescents. We report the cytomorphologic features and findings of ancillary studies of eight fine needle aspiration (FNA) biopsies from three patients (7-year-old male, 12-year-old female, 9-year-old female). Two of the biopsies suggested the initial diagnosis of PNET of the chest wall, while the remaining six documented recurrent or metastatic disease. In one of these cases the primary diagnosis made by FNA biopsy enabled the pediatric oncologists to give specific therapy for the unresectable tumor and achieve remission. Local recurrences included the chest wall (two cases), pleura (one case) and pericardium (one case), while metastatic disease involved the supraclavicular lymph node and breast. All the cases consisted of small malignant cells with a high nuclear/cytoplasmic ratio and hyperchromatic nuclei without prominent nucleoli. Homer Wright rosettes were seen in only two of the aspirates, and neuropil and ganglion cells were not present. Ancillary studies, including electron microscopy (two cases), immunocytochemistry (four aspirates from two cases) and cytogenetics (11/22 translocation, one case) performed on the aspirated material were aids in making a specific diagnosis and excluded other small round cell tumors of childhood, such as malignant lymphoma, rhabdomyosarcoma and Ewing's sarcoma. The differential diagnosis between PNET and neuroblastoma can be difficult on the basis of an FNA biopsy alone, although light microscopic morphologic differences exist. Clinical features (e.g., age, primary site, metastatic patterns), catecholamine levels, electron microscopy and cytogenetics are necessary in establishing the correct diagnosis.


Proliferative vitreo-retinal disorders: experimental models in vivo and in vitro. The aim of the present thesis was to develop, refine, and assess experimental models for the study of proliferative vitreo-retinal disorders. An intravitreal injection of a colloidal solution of microparticles was used in the primate eye to produce pathologic changes including intraocular cell invasion, cell proliferation, neovascularization, collagen synthesis, and tractional retinal detachment. In a separate primate model for laser-induced subretinal neovascularization, the origin and the occurrence of macrophages was evaluated. Examinations were performed using ophthalmoscopy, slit-lamp microscopy, light microscopy, and transmission electron microscopy. Cell cultures were employed to study the effects of vitreous humor and macrophages on the proliferation of cultured retinal pigment epithelial (RPE) cells and cultured fibroblasts using a Coulter counter. Morphologic changes were documented by phase micrography. A quantitative estimation of the extracellular matrix deposition of fibrous proteins by macrophage-modulated RPE cells as well as by vitreous-modulated RPE cells was done using enzymatic digestion and radioactive labeling techniques. A qualitative analysis of the types of collagen that was deposited in the extracellular matrices by vitreous modulated cultures was also made using indirect immunofluorescence. Using a newly developed RPE cell specific monoclonal antibody, the avidin-biotin-peroxidase labeling technique was finally employed to test the phenotypic epitope expression of macrophage-modulated and non-modulated RPE cells. A new experimental in vivo model for pathologic changes that characterize proliferative vitreo-retinal disorders was developed in the primate eye. In the model for laser-induced subretinal neovascularization, macrophages were shown to be principally recruited from the systemic circulation. Using cell cultures, it was found that both macrophage-conditioned medium and vitreous humor, separately or combined, exert mitogenic effects on RPE cells and fibroblasts. The combined effect of the two stimuli was additive, but not synergistic, on both cell lines. When incubated with macrophage-conditioned culture medium or vitreous humor, RPE cells exhibited a metaplastic transformation towards fusiform, spindle-shaped cells that were morphologically indistinguishable from fibroblasts. The extracellular matrices of RPE cells modulated by macrophage-conditioned medium also appeared converted to a more striated pattern as compared to non-modulated controls. The metaplastic transformation of cultured RPE cells reverted when experimental stimuli, macrophage-conditioned medium or vitreous humor, were withdrawn. A new in vitro method for evaluating fibrous protein deposition in the extracellular matrix by RPE cells was also described. RPE cells, that were modulated by macrophage-conditioned medium or vitreous humor, deposited less fibrous proteins per cell.(ABSTRACT TRUNCATED AT 400 WORDS)


Adhesive mechanisms of the corneal epithelium. The corneal epithelium adheres to the stroma through a series of linked structures termed collectively the adhesion complex. These structures include; intermediate filaments (keratin filaments) which are linked to the hemidesmosome; the hemidesmosome; the anchoring filaments which extend from the membrane at the hemidesmosome through the lamina lucida to the lamina densa region of the basement membrane; the anchoring fibrils which insert into the basement membrane from the stromal side; and the anchoring plaque on which anchoring fibrils terminate distal from their insertion on the basement membrane. Upon wounding, basal cells of the corneal epithelium disassemble their hemidesmosomes. During migration, the membranes along the wound bed exhibit a different kind of adhesion junction, the focal contact. This junction is present primarily in cells of the leading edge of migration and may be the provisional adhesion junction used by epithelial sheet moving to cover a wound.


Integrins as receptors for extracellular matrix proteins in human cornea. Extracellular matrix (ECM) proteins form distinct protein families that play a role during tissue maturation, wound healing and maintenance of tissue architecture. Recent studies show that there are tissue type-specific variations in their expression. ECM proteins function by complexing with each other and also by interacting with their cellular receptors, called integrins. Integrins are heterodimeric membrane glycoproteins that are partly cell type-specifically expressed in human tissues. Like other stratified epithelia, corneal epithelium expresses alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, 4, and alpha v beta 1 integrins that mediate attachment to the basement membrane and cell-cell interactions.


Integrins in the normal and healing corneal epithelium. Integrins are heterodimeric plasma membrane glycoproteins involved in cell-matrix and cell-cell interactions. The present communication reviews the distribution of several of the currently known integrin subunits in the corneal epithelium. The corneal epithelium contains the following integrin heterodimers: alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4. The expression of alpha v with an unknown beta subunit is also recognized, whereas the expression of alpha 4 and alpha 5 subunits remains controversial. Some of the changes occurring in the distribution of integrins in response to wounding will also be discussed.


Collagenolytic/gelatinolytic enzymes in corneal wound healing. We have documented changes in expression of collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in healing or ulcerating corneal wounds of rat or rabbit. Correlation of our findings with specific changes in the extracellular matrix of the repair tissue suggests two different roles for the enzymes, MMP-2 and MMP-9. MMP-2 is expressed in undamaged corneal stroma where it may degrade the occasional collagen molecule that becomes damaged. After corneal wounding, expression of this enzyme is increased and much of it appears in the active form. These changes persist for at least seven months, suggesting that MMP-2 is involved in the prolonged process of collagen remodelling in the stromal repair tissue. MMP-9 is expressed in the epithelial layer of repair tissue with a timing suggesting it might participate in controlling resynthesis of the basement membrane. MMP-9 also appears to be involved in degradation of the epithelial basement membrane that precedes corneal ulceration.


Surface-associated activation of plasminogen on gram-positive bacteria. Effect of plasmin on the adherence of Staphylococcus aureus. In this article we review a novel type of plasminogen activation on staphylococcal and streptococcal cells. The activation mechanism implies a specific binding of glu-plasminogen to bacterial surface via the lysine-binding sites of plasminogen. Association of plasminogen with bacterial surfaces greatly enhances the t-PA mediated activation which takes place only poorly in solution. The end product, surface-associated plasmin, is enzymatically active, protected against high molecular weight plasmin inhibitors and capable of converting itself from glu-plasmin to the lys-form. The modification is associated with an increased affinity of the bound lys-plasmin towards the binding molecules on bacterial surface. This novel way of retaining plasmin on the surface may be important for the bacteria to invade and penetrate surrounding tissues. Our data on the effect of plasmin on staphylococcal adherence indicate that plasmin is not very effective in cleaning bacteria from surfaces coated with extracellular matrix components, fibronectin and fibrinogen.


Experience with plasmin inhibitors.

Experience with plasmin inhibitors. The involvement and role of the plasminogen activator-plasmin system in normal and pathological wound healing is reviewed. The methods currently available for demonstrating plasmin activity are briefly described. The article also reviews some other serine proteases potentially involved in pathological wound healing processes. The current ophthalmological therapeutic measures used to regulate tissue proteolysis are also described.


Effects of growth factors on corneal wound healing. Healing of ocular surface wounds is a complex process involving migration, mitosis, and differentiation of epithelial and stromal cells. Endogenously produced peptide growth factors such as epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or transforming growth factor beta (TGF-beta) may play key roles in the natural wound healing process. Lacrimal gland cells were reported to synthesize and secrete EGF into tear fluid where it may enhance healing of corneal epithelial and stromal injuries by an exocrine pathway. EGF stimulated DNA synthesis of epithelial cells and stromal fibroblasts in culture, stimulated synthesis of fibronectin by epithelial cells and was chemotactic for human epithelial and stromal cells. Human corneal epithelial cells also synthesized TGF-alpha which may influence epithelial cells by an autocrine pathway. TGF-beta, which is a potent inducer of lysyl oxidase mRNA levels in cultures of human scleral fibroblasts, may be the factor most responsible for inducing synthesis of corneal extracellular matrix components after an injury. Treatment of epithelial injuries ocular surface wounds with exogenous peptide growth factors also accelerated healing in rabbits and primates. Treatment of severe ocular surface injuries caused by alkali with a combination of EGF, fibronectin, a synthetic collagenase inhibitor, and Aprotinin significantly blocked ulceration and enhanced epithelial regeneration. Clinical trials of topical treatment of EGF for ocular surface wounds suggest that peptide growth factors may be a valuable adjuvant for treatment of ocular surface wounds.


Reactive formation of hyaluronic acid in the rabbit corneal alkali burn. The presence and distribution of reactively formed hyaluronic acid (HA) was assessed in the rabbit cornea following a penetrating alkali burn. The injury was inflicted by applying a round, 5.5 mm, filter paper soaked in 1 N NaOH centrally on the cornea for 60 seconds. Biochemical analysis revealed a significant increase of HA two weeks after injury, a peak concentration after 1 month, and a decrease again at three months. Histochemical analysis revealed the presence of hyaluronic acid in the healing epithelium, in the repopulating keratocytes/fibroblasts, and in the cells forming the retrocorneal membrane. Extensive amounts filled lacunae in the stroma as well as the spaces between collagen lamellae. A slow restoration of normal appearing corneal stroma took place at the periphery. Significant staining for HA in lacunae was present centrally in the wound after three months.


Clinical measures to promote corneal epithelial healing. It is necessary to know the specific pathobiology of a persistent epithelial defect to determine the strategy to be employed to assist in its repair. Lid position and function must be normal and any deficiency in the quantity and quality of the tears enhanced by tear preparations and closure of the lacrimal canaliculi. Adverse drug effects must be eliminated. Multiple corneal punctures and excision of reduplicated basal lamina have virtually eliminated the problem of recurrent corneal erosions. Control of any inflammatory process also speeds healing. Vitamin supplements, especially A, reverse defects associated with xerophthalmia. In any of these diseases, mechanical treatments consisting of soft contact lenses for persistent epithelial defects and collagen shields for the delivery of antibiotics or steroids to the eye may be employed. Tarsorrhaphy relieves the problem of persistent epithelial defects in neurotrophic keratitis and a variety of other conditions characterized by persistent surface breakdown. Preliminary data from open label studies of epidermal growth factors and fibronectin are encouraging but not yet conclusive.


Surgical approaches to corneal wound healing. New approaches to improving corneal epithelial wound healing include stromal puncture and removal of abnormal basement membrane material by excimer laser ablation (phototherapeutic keratectomy). Non-healing corneal erosions and chemical burns may also benefit from laser excision of the damaged surface to permit more normal epithelial attachments. Reduction of postsurgical astigmatism may be facilitated by adjustable sutures after penetrating keratoplasty or by molding the cornea with the use of a rigid contact lens in the immediate postoperative period.


Epithelial-myoepithelial carcinoma of the parotid gland in a child. A rare case of epithelial-myoepithelial carcinoma of the parotid gland, which occurred in a child, is reported. An 8-year-old boy presented with swelling of the right parotid gland. He underwent total parotidectomy followed by irradiation for a parotid gland tumor. Three years after the operation, a recurrent tumor invading the base of the skull and the brain and metastases in the lung were noted. The patient expired in spite of extirpation of the intracranial recurrent tumor. The resected tumor showed a characteristic histologic feature: double-layered tubular structures composed of inner dark cells (epithelial cells) and outer clear cells (myoepithelial cells). This patient may be the youngest one with the epithelial-myoepithelial carcinoma reported in the literature.


Comparison of the effect of a mitochondrial uncoupler, 2,4-dinitrophenol and adrenaline on oxygen radical production in the isolated perfused rat liver. Using the isolated perfused rat liver, we examined the effect of stimulation of mitochondrial respiration by 2,4-dinitrophenol (2,4-DNP) and adrenaline on reactive oxygen species (ROS) production, liver damage and lipid peroxidation. ROS production was monitored by luminol- and lucigenin-enhanced chemiluminescence and oxygen uptake was measured simultaneously. Liver damage and lipid peroxidation were evaluated by measuring hepatic lactate dehydrogenase (LDH) and thiobarbituric acid reacting substances (TBARS) release. Tissue ROS level decreased and oxygen uptake increased soon after 2,4-DNP infusion. On termination of 2,4-DNP infusion, there was a sharp increase in lucigenin-enhanced chemiluminescence, which declined slowly, but luminol-enhanced chemiluminescence did not change prominently. Hepatic LDH and TBARS release increased gradually during 2,4-DNP infusion and were manifested by termination of the infusion. Allopurinol did not affect ROS production and TBARS release, but delayed increases in LDH release after termination of 2,4-DNP infusion. Adrenaline, which stimulates mitochondrial respiration without uncoupling caused similar but smaller ROS changes observed in 2,4-DNP. LDH and TBARS release were not affected significantly by adrenaline infusion. These results indicate that uncoupling of oxidative phosphorylation decreases ROS production and restoration of oxidative phosphorylation enhances ROS production and liver damage. Xanthine oxidase is unlikely to contribute to enhanced ROS production after termination of 2,4-DNP but has some protective effect during uncoupling.


2-Amino-3-phosphonopropionate fails to block postsynaptic effects of metabotropic glutamate receptors in rat hippocampal neurones. Metabotropic glutamate receptors (mGluRs) operate via the phosphoinositide second messenger cascade and have various modulatory effects on central neurones. 2-Amino-3-phosphonopropionate (AP3) has been proposed as a selective antagonist of mGluR and used to explore the physiological functions of mGluR. We have compared the effects of mGluR agonists in the presence and absence of AP3 using intracellular recording from CA1 pyramidal neurones in rat hippocampal slices. Two mM D,L-AP3 or 1 mM L-AP3 did not cause any detectable change in the effects of mGluR agonists trans-1-amino-cyclopentyl-1,3-dicarboxylate (t-ACPD) or quisqualate. These agonists still induced depolarization, inhibition of the slow after-hyperpolarization and slowing of the spike repolarization. The results argue against AP3 being an antagonist of postsynaptic mGluRs.


Adrenocorticoid regulation of Na+,K(+)-ATPase in adult rat kidney: effects on post-translational processing and mRNA abundance. The mechanisms by which adreno-corticoid hormones regulate Na+,K(+)-ATPase in adult kidney were studied in adrenalectomized (Adx) rats. Five days after adrenalectomy, Na+,K(+)-ATPase activity was significantly reduced in the renal cortex homogenate (C = 13.0 +/- 0.8 vs. Adx = 7.1 +/- 0.7 mumol Pi mg-1 protein h-1) and in renal microsomes (C = 30.3 +/- 1.9 vs Adx = 14.6 +/- 1.3 mumol Pi mg-1 protein h-1). Glucocorticoid replacement treatment of adrenalectomized rats with betamethasone (20 micrograms kg-1 body wt twice daily for 5 days) effectively counteracted the observed reduction in Na+,K(+)-ATPase activity. In cortical homogenate the protein level of alpha 1 and beta 1 subunits measured in immunoblots was not significantly different in Adx and control rats, indicating that 5 days after adrenalectomy the alpha 1 and beta 1 subunits were present in renal cortical cells to almost normal extent but could not be assembled into a transmembrane functional unit. In support of this conclusion we found that the protein level of both the alpha 1 and beta 1 subunits was significantly lower (P less than 0.001 for both subunits) in microsomes from Adx than in control rats. The mRNA abundance for alpha 1 and beta 1 subunits were not lower in Adx as compared to control rats 1 and 5 days after surgery. However, if Adx rats were given a single dose of betamethasone (600 micrograms kg-1 body wt), a significant 2-fold increase in both alpha 1 and beta 1 mRNAs was observed (P less than 0.05 for both subunits).(ABSTRACT TRUNCATED AT 250 WORDS)


Calcium supplementation and thyroid hormone protect against gentamicin-induced inhibition of proximal tubular Na+,K(+)-ATPase activity and other renal functional changes. Gentamicin can cause proximal tubule necrosis. We have shown that inhibition of PT Na+,K(+)-ATPase activity is rapidly induced by gentamicin. We have now investigated whether manipulations known to attenuate the negative effects of gentamicin on renal excretory capacity, i.e. high calcium intake and L-thyroxine treatment, will also attenuate gentamicin-induced inhibition of Na+,K(+)-ATPase activity and ameliorated signs of proximal tubule damage. Rats were gentamicin- or vehicle-treated for 7 days. Sub-groups were given 4% calcium (Ca) supplements or L-thyroxine 20 micrograms 100 g-1 body weight daily. Gentamicin significantly reduced the glomerular filtration rate and increased the urinary excretion of the proximal tubule lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. Gentamicin significantly reduced proximal tubule Na+,K(+)-ATPase activity, measured in single permeabilized proximal tubule segments. Sodium excretion was inversely correlated to proximal tubule Na+,K(+)-ATPase activity. Both calcium and L-thyroxine alleviated all gentamicin-induced side-effects on renal function as well as on proximal tubule Na+,K(+)-ATPase activity. Calcium and L-thyroxine had no significant effect on renal function. L-thyroxine, but not calcium, increased proximal tubule Na+,K(+)-ATPase activity in control rats. Renal cortical tissue gentamicin concentration was not influenced by calcium but was significantly lowered by L-thyroxine. Two procedures which, via different mechanisms, afford protection from gentamicin-induced changes in renal function also give protection from gentamicin-induced inhibition of Na+,K(+)-ATPase activity. This suggests that loss of integrity of the Na+,K(+)-ATPase enzyme contributes to gentamicin-induced nephrotoxicity.


Calcium-induced net potassium uptake of pig hearts in vivo. To determine whether the catecholamine-induced myocardial potassium uptake could be mimicked by increasing extracellular and intracellular calcium concentrations in vivo, we measured changes in myocardial potassium balance in nine anaesthetized open-chest pigs with PVC-valinomycin electrodes in arterial and coronary sinus blood. CaCl2 infusion (200-400 mumol min-1) into the left coronary artery increased coronary sinus blood calcium concentration from 2.29 (2.19-2.42) to 4.63 (3.76-5.67) mmol l-1 (median, 95% confidence interval, P = 0.01) indicating a similar increment in myocardial extracellular calcium concentration. The contractility measure LV dP/dt increased 95 (76-147) %, indicating a substantial increment in intracellular calcium concentration. During the CaCl2 infusion coronary sinus potassium concentration declined to a nadir 0.12 (0.09-0.17) mmol l-1 below baseline (P = 0.008) whereas arterial concentration remained unchanged. Peak myocardial potassium uptake was 18 (7-32) mumol min-1 100 g-1 and occurred 150 (110-195) s after start of infusion. The response remained unaltered after adrenoceptor blockade by prazosin and propranolol. Prolonged CaCl2 infusion caused a net myocardial potassium loss which was accompanied by metabolic and haemodynamic indications of myocardial ischaemia. These findings are consistent with enhanced Na-K pump activity in the intact beating pig heart in response to increased extracellular and intracellular calcium concentrations.


[Benign fibrous histiocytoma of the female urethra]. Presentation of one case of benign fibrous histiocytoma of the female urethra in a 49-year old woman. Investigation of the various clinical and diagnostic aspects. Vaginal tumorectomy was curative, and no recurrence was observed 14 months after the control.


Contributions to arachidonic acid release in mouse cerebrum by the phosphoinositide-phospholipase C and phospholipase A2 pathways. Recent studies have indicated two major mechanisms for the release of arachidonic acid (20:4) from membrane phospholipids: 1) activation of phospholipase A2 and 2) stimulated hydrolysis of poly-phosphoinositides (PI) and diacylglycerols (DG) through phospholipase C and diacylglycerol lipase, respectively. In mammalian brain both mechanisms seem to be operable, although the relative contributions by these two pathways have not been carefully assessed. In this study three experimental protocols were used to examine 20:4 release in brain due to ischemia and agonist stimulation, as well as the metabolic relationship between this release and the increase in diacylglycerols, lysophospholipids, and inositol phosphates. The preferential release of arachidonic acid during the initial phase after decapitation was attributed mainly to the sequential hydrolysis of poly-PI to DG. During the second phase, the release of 20:4 along with other free fatty acids (FFA) correlated well with the increase in labeled lysophospholipids, suggesting the involvement of phospholipase A2. Diacylglycerols in brain are enriched in 18:0 and 20:4. Decapitation induced a rapid increase in the level of DG, which remained elevated during the 30 min period under study. Between 5 sec and 5 min, the increase in FFA lagged behind that of DG. The parallel increases in 18:0 and 20:4 in the FFA pool further support the notion that, during the early phase, 20:4 could be derived from the sequential hydrolysis of poly-PI and DG. Decapitation also induced a sequential appearance of Ins(1,4,5)P3, Ins(1,4)P2, and Ins(4)P, which peaked at 30 sec, 1 min, and 2 min, respectively. The level of 20:4 in brain was also examined with respect to poly-PI turnover due to stimulation by cholinergic agonists. Administration of pilocarpine to lithium-treated mice resulted in increased accumulation of labeled inositol monophosphate (IP1) compared to the amount in controls receiving lithium alone, as well as a less obvious increase in 20:4. Both pilocarpine-mediated increases (IP1 and 20:4) could be blocked by atropine. These results point to the presence of an active mechanism for poly-PI turnover and for the recycling of 20:4 in brain.


Non-eicosanoid functions of essential fatty acids: regulation of adenosine-related functions in cultured neuroblastoma cells. Studies have demonstrated that augmenting the omega 6 polyunsaturated-fatty-acid (PUFA) content of N1E-115 neuroblastoma cells by media supplementation with linoleic acid results in greater than or equal to 2-fold increases in basal levels of intracellular cyclic AMP (cAMP). Data suggested some involvement of increased production of adenosine from endogenous metabolites; however, increases in adenosine were not related to increased activity of 5'-nucleotidase or decreased uptake of extracellular adenosine. PUFA-dependent elevations in basal cAMP were evident within 1 min of exposure to a phosphodiesterase inhibitor; this phenomenon did not appear to be due to PUFA-dependent changes in Ca2+ uptake or to increases in sensitivity of adenylate cyclase to Ca2+. Forskolin-stimulated cAMP formation was 3-fold higher in PUFA-enriched cells than in control cells, which suggested a direct effect on the functioning of the catalytic unit. Linoleic acid supplementation resulted in a 2-fold increase in the maximum amounts of cAMP produced in response to the stable adenosine analogue, 5'-N'ethylcarboxy-amidoadenosine (NECA). The altered stimulatory response did not involve eicosanoid formation, but may have been related to an increase in the number of stimulatory adenosine receptors, as judged by binding of [3H]NECA. These studies indicate that membrane PUFA modulate adenosine-related functions in neuroblastoma cells, and suggest that a complex series of mechanisms is involved in this regulation.


Adjunctive therapy with low molecular weight heparin with recombinant tissue-type plasminogen activator causes sustained reflow in canine coronary thrombosis. Rethrombosis of the infarct-related artery after pharmacologic thrombolysis is a major limitation of the thrombolytic therapy. Platelet and fibrin deposition in the coronary artery after recombinant tissue-type plasminogen activator (rTPA) may play a leading role in reformation of thrombus. Therefore we examined the effect of low molecular weight heparin (LMWH) as adjunctive treatment with rTPA in a dog model of electrically induced intracoronary thrombus. Fourteen dogs, in which a stable coronary thrombus was induced with delivery of 100 microA of anodal direct current, were randomly given an intravenous bolus of LMWH, 75 IU/kg (n = 6), or saline (n = 8), followed by intravenous rTPA, 1 mg/kg over 20 minutes. LMWH (75 IU/kg) or saline was continuously infused over 90 minutes after rTPA-induced thrombolysis. Reperfusion occurred at 29 +/- 7 minutes in six of eight dogs receiving rTPA plus saline (reperfusion rate 75%), while reperfusion occurred at 18 +/- 3 minutes in all six dogs receiving rTPA plus LMWH (both p = NS versus rTPA plus saline group). Coronary reocclusion occurred in 83% of dogs given rTPA plus saline, but only in one dog (17%) given rTPA plus LMWH (p less than 0.05). Magnitude of reflow at 60 minutes of reperfusion was higher in the rTPA plus LMWH group than in the rTPA plus saline group (51 +/- 14 ml/min versus 10 +/- 9 ml/min; p less than 0.05). As expected, partial thromboplastin time was greater in rTPA plus LMWH than in rTPA plus saline-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Existence of PAF receptors in human platelets and human lung tissue but not in the human myocardium. The aim of this study was to investigate whether platelet-activating factor (PAF), PAF receptors, and PAF receptor-mediated effects in the human myocardium play a role in cardiac depression during anaphylaxis or septic shock. The effects of PAF, the biologically inactive derivative lyso-PAF, and the specific PAF antagonist WEB 2086 were studied in human myocardial tissue, in human coronary arteries, in human platelets, and in human lung tissue. PAF (C16-PAF, C18-PAF; 0.000001 to 1 mumol/L) had no effect on isometric force of contraction of electrically driven right atrial trabeculae (patients undergoing aortocoronary bypass surgery) and left ventricular papillary muscle strips (mitral valve replacement). PAF (0.2 mumol/L) did not influence the concentration-response curve of either the beta-adrenoceptor agonist isoprenaline (ISO, 0.0001 to 1 mumol/L) or the m-cholinoceptor agonist carbachol (CARB, 0.0001 to 10 mumol/L). The effectiveness (ISO +4.7 +/- 0.7 mN, PAF + ISO + 4.3 +/- 0.44 mN, CARB -2.7 +/- 1.06 mN; PAF + CARB -2.6 +/- 0.52 mN) and the potency--as indicated by the EC50 values--of both isoprenaline and carbachol were identical with and without pretreatment with PAF (0.2 mumol/L). PAF at concentrations of 0.000001 to 10 mumol/L exerted no effect on force of contraction either precontracted (prostaglandin F2 alpha, 0.3 mumol/L) or unprecontracted in human coronary artery rings. Histamine (0.01 to 100 mumol/L) and noradrenaline (0.001 to 30 mumol/L) initiated concentration-dependent contraction in human coronary artery rings (EC50: histamine, 1.86 mumol/L; noradrenaline, 0.69 mumol/L). At lower concentrations (PAF, 0.01 mumol/L) PAF produced complete aggregation of human platelets. In human platelet membranes and lung membranes, 3H-WEB 2086 exhibited saturable high-affinity binding (KD 14.4 nmol/L and 14.3 nmol/L). The maximal binding capacity was 292 fmol/mg protein and 268 fmol/mg protein, respectively. In displacement experiments PAF (0.01 to 10000 nmol/L) and WEB 2086 (0.01 to 10000 nmol/L), but not lyso-PAF, completely displaced 3H-WEB 2086 from its binding sites on human and lung membranes. In contrast, neither in left ventricular membranes nor in right atrial membranes was specific binding of 3H-WEB 2086 observed. These results suggest that there are neither specific PAF receptors nor direct PAF-mediated actions in human myocardial tissue or human coronary artery rings. The effects of PAF on myocardial function may be due to the activation of mediators (e.g., histamine).


Effect of immunostimulant therapy on acute viral myocarditis in an animal model. The effect of immunostimulant therapy on acute viral myocarditis, which was induced with encephalomyocarditis virus, was investigated in 4-week-old male BALB/c mice. In vitro, peritoneal exudate cells and spleen cells from mice that were pretreated with a synthetic immunoactivating peptide, FK565 (heptanoyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alan ine), significantly inhibited the multiplication of encephalomyocarditis virus in BALB/c 3T3 cells compared with peritoneal exudate cells and spleen cells from untreated mice (6.14 +/- 0.21 log10 plaque-forming units (pfu) per milliliter, control: 6.59 +/- 0.03, p less than 0.05; 3.55 +/- 0.23, control: 5.64 +/- 0.09, p less than 0.01, respectively), although FK565 did not inhibit viral replication directly. The mice were inoculated intraperitoneally with 100 pfu of encephalomyocarditis virus. FK565 (1 mg/kg/day), which was administered 1 day before or simultaneously with virus inoculation, effectively inhibited myocardial viral replication (2.77 +/- 0.17 log10 pfu/mg, 2.46 +/- 0.35, log10 pfu/mg, respectively, control: 3.33 +/- 0.26, p less than 0.025) and increased survival (70% and 60%, respectively, control: 20%, p less than 0.01). Histopathologic findings were scored on a scale of 0 to 4. Treatment with 1 mg/kg/day of FK565 that was started 1 day before virus inoculation was most effective in reducing the inflammatory response (1.2 +/- 0.63, control: 2.0 +/- 0.81, p less than 0.05) and myocardial necrosis (1.2 +/- 0.42, control: 2.0 +/- 1.00, p less than 0.025). The present study suggests that immunostimulant therapy improves the course of viral myocarditis during the virus-mediated phase.(ABSTRACT TRUNCATED AT 250 WORDS)


Psyllium husk. I: Effect on plasma lipoproteins, cholesterol metabolism, and atherosclerosis in African green monkeys. Psyllium's effects on plasma and lipoprotein cholesterol concentrations, cholesterol metabolism, and diet-induced atherosclerosis were studied in adult male African green monkeys (Cercopithecus aethiops). Animals were fed for 3.5 y one of three experimental diets: low-cholesterol cellulose (LCC), high-cholesterol cellulose (HCC), or high-cholesterol psyllium (HCP). The LCC and HCP groups had significantly (P less than 0.05) lower plasma cholesterol concentrations (39% lower) at 1 mo than did the HCC group. These responses persisted throughout the study. Plasma cholesterol changes were due to a reduction in intermediate-density and low-density lipoproteins; very-low and high-density-lipoprotein concentrations were similar among groups. Aortic atherosclerosis, evaluated as percent sudanophilia at 3.5 y, was lowest in the LCC group, intermediate in the HCP group, and highest in the HCC group. Cholesterol absorption, neutral steroid and fat excretion, HMGCoA reductase activity (in intestine and liver), and body weight were unrelated to psyllium's hypocholesterolemic effects.


Psyllium husk. II: Effect on the metabolism of apolipoprotein B in African green monkeys. Dietary psyllium's ability to reduce low-density-lipoprotein (LDL) cholesterol is presumably mediated by increased LDL catabolism and/or reduced LDL synthesis. To distinguish between these possibilities, apolipoprotein B (apo B) metabolism was studied in adult male African green monkeys consuming one of three semipurified diets: low-cholesterol cellulose (LCC), high-cholesterol cellulose (HCC), or high-cholesterol psyllium (HCP). 131I-labeled LDL and 125I-labeled VLDL were injected simultaneously into animals; blood samples were drawn at selected times and apo B specific activity determined in VLDL, IDL, and LDL. Based on a multicompartmental model, LDL apo B pool size and de novo apo B transport were elevated significantly in HCC animals compared with HCP and LCC animals. Differences in LDL transport, although not significant, paralleled differences observed in LDL apo B pool size. Fractional catabolic rates were similar among groups (HCC 0.040 +/- 0.010; HCP 0.042 +/- 0.009, and LCC 0.043 +/- 0.004 pools/h). These data suggest that dietary psyllium reduces plasma cholesterol concentrations by decreasing LDL synthesis.


Oat bran increases serum acetate of hypercholesterolemic men. Mechanisms for the hypocholesterolemic effects of oat bran remain unclear. Soluble fibers such as oat bran are fermented in the colon to short-chain fatty acids (SCFAs), which may enter the portal vein and attenuate hepatic cholesterol synthesis. To compare effects of oat bran and wheat bran on serum SCFA concentrations, 20 hypercholesterolemic men entered a metabolic ward and received control diets for 1 wk followed by oat-bran or wheat-bran diets for 3 wk. Oat bran decreased serum cholesterol 12.8% (P less than 0.001) whereas wheat bran had no effect. Peripheral serum SCFA concentrations were measured seven times over 14 h at the end of each diet. Serum acetate values from 1200 to 2200 were significantly higher in subjects fed oat-bran vs wheat-bran diets. Peak and incremental peak acetate values were also significantly higher than control values in subjects fed oat bran but not in subjects fed wheat bran. SCFA responses may contribute to the hypocholesterolemic effects of oat bran.


Contractile agonists activate voltage-dependent calcium channels in airway smooth muscle cells. To determine whether agents that cause contraction of airway smooth muscle affect sarcolemmal calcium channel activity, unitary calcium channel currents (using Ba2+ as the charge carrier) were recorded in on-cell configuration from acutely dissociated (dog, pig, and ferret) and cultured (human) airway smooth muscle cells. Addition of the contractile agonists methacholine or bradykinin increased the open-state probability of the large-conductance calcium channel 37.2- and 45-fold, respectively. The increase in open-state probability was not due to cellular depolarization because increases occurred in the absence of depolarization. Channel activation by the agonist was determined to result in the favoring of a long (16.5 +/- 5.0 ms) open lifetime for the channel, which was not observed under control conditions, in the absence of BAY K 8644. We also report the unitary calcium channel currents from a second, smaller conductance calcium channel. This channel was present in all cell types and had a mean conductance of 9.5 +/- 0.8 pS (80 mM Ba2+). Exposure of cells to agonist also resulted in an increase in the open-channel probability of the small-conductance calcium channel (10.4-fold), which did not result from cellular depolarization. These experiments demonstrate that the molecular pathways exist between contractile agonist receptors and sarcolemmal calcium channels in airway smooth muscle cells. Because membrane patches were not directly exposed to agonist, receptor-channel linkage probably occurs via a second messenger-coupling pathway.


Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic beta-cells. It has been proposed that certain cytokines secreted by islet-infiltrating leukocytes may be involved in the pathogenesis of insulin-dependent diabetes mellitus by participation in beta-cell destruction. In the present study, the impact of various cytokines on replication and long-term insulin secretion by pancreatic beta-cells was investigated. To this end, fetal rat pancreatic islets containing a high fraction of beta-cells were exposed in culture for 1-3 days to interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interferon-alpha (IFN-alpha), and interleukin-6 (IL-6) at different concentrations. It was found that IL-1 beta markedly decreased beta-cell DNA synthesis during the first day of exposure, an effect that vanished after 2 days and was turned into a potent and dose-dependent stimulation by 3 days of exposure. At this latter time point, IL-1 beta also amplified the mitogenicity of growth hormone (GH) and 16.7 mM glucose. In contrast, basal as well as glucose- and GH-stimulated insulin secretion was consistently suppressed by IL-1 beta from days 1-3. IL-1 beta also lowered the islet adenosine 3',5'-cyclic monophosphate (cAMP) content at all time points studied. However, addition of the stimulatory cAMP analogue Sp-diastereomer of adenosine 3',5'-cyclic monophosphothioate or pertussis toxin, which themselves enhanced DNA synthesis and insulin secretion, failed to prevent the inhibitory actions of IL-1 beta on these parameters, making it unlikely that a decrease in cAMP is an important event in transduction of the inhibitory effects of the cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)


Alpha-subunits of Gs and Gi in adipocyte plasma membranes of genetically diabetic (db/db) mice. The adipocyte membrane G protein pattern, beta-adrenergic receptor activity, and adenylyl cyclase were determined in adipocyte membranes of the db/db mouse, a mutant that is a model of diabetes preceded by hyperinsulinemia, hyperglycemia, and extreme obesity. These studies were undertaken to determine whether the alterations already noted in the ob/ob mouse and those in the db/db mouse are similar and related to the hormonal defects, particularly the hyperinsulinemia and hyperglycemia prevalent in these animals (cf. Ref. 11). The ADP ribosylation data show that Gs alpha was more highly labeled in the tissues of the db/db mutant than in the homozygous control, but there was no significant difference in the amount of ADP-ribose incorporated in the Gi alpha-subunits. Quantification of the proteins by immunodetection revealed that the long (46-kDa) form of Gs alpha was significantly less abundant in the db mutant than in its control, whereas there was no difference in the short (42-kDa) form. Gi alpha-peptides corresponding to Gi alpha 2 and Gi alpha 1 were both less abundant in the db mutant than in the homozygous control. These data contrasted with those obtained for ob mutants and their lean homozygous controls reported previously (4) and confirmed here. It is concluded on the basis of these studies that factors other than the hormonal status are responsible for the G protein patterns in the ob and db mutants. Differences in G protein patterns noted in between the control groups (B/Ks or B/6 homozygotes) correlated strongly with the quantitative differences in adenylyl cyclase response in the two strains.(ABSTRACT TRUNCATED AT 250 WORDS)


cAMP-dependent protein kinase mediates hydrosmotic effect of vasopressin in collecting duct. The role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in mediating the hydrosmotic effect of vasopressin in in vitro microperfused rabbit cortical collecting ducts (CCDs) was examined. We measured PKA substrate phosphorylation and water permeability [hydraulic conductivity (Lp) = 10(-7) cm.atm-1.s-1], stimulated by substituted cAMP analogues selective for a unique cAMP binding site (site A or B) on PKA regulatory subunit (R). Synergy between site A- and site B-selective analogues suggests involvement of PKA, because both sites must be occupied for R to dissociate from the catalytic subunit (C), allowing phosphorylation to proceed. As single agents, the site B-selective analogues 8-(4-chlorophenylthio)-cAMP (8-CPT) and 8-thiomethyl-cAMP (8-SCH3) were at least two orders of magnitude more potent than the site A-selective analogues N6-monobutyryl-cAMP (N6-mono) or N6-benzoyl-cAMP (N6-benz). Combinations of subthreshold concentrations of two site A analogues (N6-mono+N6-benz) or two site B-selective analogues (8-CPT + 8-SCH3) failed to significantly increase protein phosphorylation or water permeability. In contrast, combination of a site A plus site B analogue synergistically stimulated both protein phosphorylation and Lp. Rp-cAMPS, an inhibitor of cAMP binding to PKA, reduced both vasopressin (41% inhibition)- and cAMP (56% inhibition)-stimulated water permeability. H-89 (50 microM), an inhibitor of PKA kinase activity, also blocked cAMP-stimulated water permeability (90% inhibition). These findings suggest that vasopressin-induced water permeability in the rabbit CCD is mediated by PKA.


Regulation of an epithelial chloride channel by direct phosphorylation and dephosphorylation. A native chloride channel in Necturus gallbladder epithelial cells is opened by a theophylline-induced rise in cellular cyclic AMP and is closed by removal of theophylline or by addition of specific antibody; however, it does not close if okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is added. The purified channel reconstituted into lipid bilayers closes upon the addition of protein phosphatase 2A and is reopened by the addition of Mg-ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. These results indicate that the channel protein is purified in a phosphorylated state and that its functional characteristics are at least partly controlled by direct phosphorylation and dephosphorylation.