Rett syndrome: stimulation of endogenous biogenic amines. Transient hypercapnic hyperoxemia was induced in two Rett syndrome children by the administration of a gaseous mixture of 80% O2 and 20% CO2. Time course studies of neurotransmitters and their metabolites showed an immediate and marked increase in central biogenic amine turnover following inhalation of the gas mixture. The increased turnover of biogenic amines was associated with improved clinical changes. This suggests a coupled relationship and provides further support for an etiological role of neurotransmitter dysfunction in Rett syndrome. In a complementary study, elevation of pulmonary CO2 by application of a simple rebreathing device resulted in improvement of abnormal blood gases and elimination of the Cheyne-Stokes-like respiratory pattern of the Rett syndrome. Near normalization of the EEG occurred when a normal respiratory pattern was imposed by means of a respirator. Taken together, these results lead to the preliminary conclusion that cerebral hypoxemia secondary to abnormal respiratory function may contribute to diminished production of biogenic amines in Rett syndrome.


Enhancement of extracellular protein concentrations during long-term potentiation in the rat hippocampal slice. The possibility that the enhancement of extracellular protein concentrations during long-term potentiation is related to the maintenance of long-term potentiation was examined in area CA1 of rat hippocampal slices. First, we found that during the 3 h after induction, long-term potentiation maintenance was correlated with a persistent enhancement of extracellular protein concentrations. Second, when the protein synthesis inhibitor cycloheximide was applied 10-15 min before long-term potentiation induction, the drug blocked both the maintenance of long-term potentiation and the elevation of extracellular protein concentrations. These results suggest that the elevation of extracellular protein concentrations requires new protein synthesis. The results further indicate that the newly synthesized proteins may play a role in the maintenance of long-term potentiation.


Actions of acetylcholinesterase in the guinea-pig cerebellar cortex in vitro. Acetylcholinesterase is released in a calcium-dependent manner when afferents of the cerebellar cortex are stimulated. Since cholinergic transmission is probably insignificant in the cerebellar cortex, the esterase itself might serve as a transmitter or modulator. Therefore, the effect of acetylcholinesterase in the cerebellum was investigated in slices of guinea-pig cerebella during intracellular recording from Purkinje cell somata or dendrites. Addition of acetylcholinesterase (20 U/ml) to the superfusion medium did not change the membrane potential or the input resistance of the Purkinje cells. Thus, esterase does not act like a classical transmitter. The threshold for Na+ spikes generated by intracellular current injection was unaffected, but the threshold for Ca2+ spikes was increased. This increase was abolished by tetrodotoxin (1 microM). Furthermore, when Ca2+ currents were blocked by substituting Mn2+ for Ca2+ (2 mM) a decrease in a Na+ plateau potential was seen in the presence of esterase. The effect of acetylcholinesterase of Ca2+ spikes is therefore most likely due to a reduction of the non-inactivating Na+ current of the Purkinje cell membrane. When present this current contributes to activation of Ca2+ spikes in dendrites. Acetylcholinesterase also enhanced the response of Purkinje cells to the excitatory amino acids glutamate and aspartate thought to be transmitters in the cerebellar cortex. The responses became larger and faster in the presence of esterase. Responses to climbing fibre stimulation were also enhanced by acetylcholinesterase. The late part of this synaptic response was increased. The potentiation by esterase of responses of Purkinje cells to excitatory amino acids and to climbing fibre stimulation may be mediated through interference with transmitter uptake, because it was prevented by treatment with DL-2-amino-4-phosphonobutyric acid (0.5 mM) and di-hydrokainate (0.1 mM). None of the effects of esterase was due to hydrolysis of acetylcholine because irreversible inhibition of the catalytic site of the enzyme with soman did not prevent the actions. The observations were specific for acetylcholinesterase. Butyrylcholinesterase (20-40 U/ml) showed none of the effects. It is concluded that acetylcholinesterase in the cerebellar cortex seems to mediate a novel type of modulation by two separate mechanisms. Esterase reduces the tendency towards Ca2+ spike generation in Purkinje cells. Ca2+ spikes are followed by afterhyperpolarizations and in their absence firing of Na+ spikes at higher frequencies is possible. Secondly, there is an enhancement of the action of excitatory transmitters so that the extended operating range can be utilized.


Extracellular dopamine in striatum: influence of nerve impulse activity in medial forebrain bundle and local glutamatergic input. Microdialysis probes were used to measure dopamine in, and to administer glutamate receptor antagonists and agonists to, the striatum of unanesthetized rats. Antagonists used were: kynurenate, 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione. Agonists used were: N-methyl-D-aspartate and kainate. In some rats an additional dialysis probe was implanted in medial forebrain bundle for infusion of tetrodotoxin (10 microM) to block action potential propagation along dopaminergic axons in this pathway. The latter treatment reduced dopamine in striatal dialysate to below detectable levels (less than 0.5 pg). The quantity of dopamine in striatal dialysate was not reduced by the local application of glutamate receptor antagonists. At lower concentrations, the receptor antagonists failed to alter significantly the quantity of dopamine, whereas the highest concentration of each antagonist increased the amount of dopamine in the dialysate. At the highest concentration tested (0.75 mM or 1.0 mM), as well as at a lower concentration (0.1 mM), 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione blocked the dopamine-releasing effects of exogenously applied N-methyl-D-aspartate (1.0 mM) or kainate (0.1 mM), respectively. Thus, concentrations of glutamate receptor antagonists that produced effective pharmacological blockade of the respective receptors had no effect on the basal amount of dopamine in striatal extracellular fluid. Finally, N-methyl-D-aspartate and kainate produced a significant elevation in extracellular dopamine during the infusion of tetrodotoxin into the medial forebrain bundle, indicating that impulse activity in this pathway is not necessary for dopamine release produced by glutamate receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)


Aminopeptidases in the circumventricular organs of the mouse brain: a histochemical study. The localization of four membrane-bound aminopeptidases--aminopeptidase A, aminopeptidase M, dipeptidylpeptidase IV, and gamma-glutamyl transpeptidase--known as characteristic enzymes of the blood-brain barrier was studied in the microvasculature of some circumventricular organs of the mouse brain (subfornical organ, area postrema, choroid plexus, and neurohypophysis). Enzyme activities were demonstrated histochemically in chloroform-acetone-pretreated cryostat sections applying an azo-coupling method. Reactions were evaluated using light microscopy and end-point microdensitometry. The results revealed differences in microvascular enzyme pattern between circumventricular organs and regions having a blood-brain barrier. Moreover, the cytochemical picture of the circumventricular organs themselves was not uniform. Dipeptidylpeptidase IV reaction showed a strongly reduced activity in the microvessels of all studied circumventricular organs. On the other hand, aminopeptidase M seemed to be present in both the leaky and the tight capillaries. Only a low activity of aminopeptidase A was found in parts of the choroid endothelium and the subfornical organ microvasculature. gamma-Glutamyl transpeptidase could neither be detected in the capillary part of the choroid plexus nor in the neurohypophysis. We are led to conclude that at least dipeptidylpeptidase IV might be involved in special mechanisms of the blood-brain barrier.


Striatal monoamine neurotransmitters and metabolites in dominantly inherited olivopontocerebellar atrophy. We measured the levels of the monoamine neurotransmitters and metabolites in striatum of 14 patients with end-stage dominantly inherited olivopontocerebellar atrophy (OPCA). On average, dopamine levels were reduced in putamen (-53%), caudate (-35%), and nucleus accumbens (-31%). However, individual patient values showed a wide variation, indicating that mild to moderate striatal dopamine loss is a common but not constant feature of OPCA. Seven patients had marked putamen dopamine loss (-62% to -81%) but without evidence of correspondingly severe substantia nigra cell damage; this suggests the possibility of a "dying-back" phenomenon in which nerve terminal loss precedes cell body degeneration. Severe substantia nigra cell loss with almost total (-95% to -99%) putamen and caudate dopamine depletion was present in two patients; however, none of the 14 patients had had a clinical diagnosis of parkinsonism or was receiving antiparkinsonian medication. Mean striatal serotonin levels were normal, whereas concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were elevated by 47% to 63%; this suggests increased activity of raphe dorsalis serotonin neurons innervating the striatum, which might aggravate the functional consequences of the dopamine deficit.


N-acetyl-L-aspartate and other amino acid metabolites in Alzheimer's disease brain: a preliminary proton nuclear magnetic resonance study. We used proton nuclear magnetic resonance spectroscopy in this preliminary study of perchloric acid extracts of 12 Alzheimer's disease (AD) and five control brain samples to measure the relative levels of taurine, aspartate, glutamine, glutamate, gamma-aminobutyric acid (GABA), and the putative neuronal marker, N-acetyl-L-aspartate (NAA). We found no significant changes in taurine, aspartate, or glutamine. NAA was lower in AD compared with control, and this decrease correlated with the number of senile plaques and neurofibrillary tangles in adjacent tissue sections. GABA levels also were lower in AD brain. Glutamate levels were greater in AD than control and showed a close, inverse correlation with NAA levels. These findings suggest that the decrease in NAA reflects neuronal loss and that remaining neurons could be exposed to a relative excess of glutamate and a relative lack of GABA. If present in the neurotransmitter pool, this imbalance could result in neurotoxic cell damage. This hypothesis is further supported by in vitro and in vivo phosphorus 31 nuclear magnetic resonance findings.


Pretreatment with NMDA antagonists limits release of excitatory amino acids following traumatic brain injury. After central nervous system (CNS) trauma, there are marked elevations in the extracellular levels of excitatory amino acids (EAA), which are believed to contribute to delayed tissue damage. Administration of N-methyl-D-aspartate (NMDA) receptor antagonists reduces injury severity after brain or spinal cord trauma, presumably by blocking the postsynaptic NMDA receptor. In the present studies, levels of extracellular amino acids were monitored by microdialysis during, and after, a moderately severe fluid-percussion brain injury to rats. Pretreatment (15 min prior to injury) with the non-competitive NMDA antagonist dextrorphan or the competitive NMDA antagonist CGS 19755 significantly attenuated the post-traumatic increase in extracellular glutamate. Pretreatment with dextrorphan attenuated the post-traumatic increase in extracellular levels of aspartate; although these differences did not reach significance when examined as absolute values, they were significant when analyzed as percent increase over pre-trauma baseline levels. These results are consistent with recent experiments and suggest that NMDA antagonists may limit the release of glutamate and aspartate after trauma through a presynaptic mechanism.


[Effect of psychotropic drugs on vigilance and motor performances]. This review paper deals with the impact of psychotropic drugs on vigilance, awakening and motricity. Antidepressants can be divided into 3 categories, depending on the subject's awakening: sedatives with a strong anticholinergic component, compounds devoid of positive or negative impact on cognition and stimulating antidepressants. The principal effect of lithium is to lengthen the reaction time. Taken acutely, neuroleptics produce alterations of fine motor gestures, but when taken chronically they spare the functioning of cognition. Benzodiazepines act on vigilance in various ways, depending on their half-life and on their plasma peak time after oral administration. The effect of anticonvulsants on cognition is more pronounced with phenytoin and barbiturates than with carbamazepine or valproate sodium. The problems of comparative analysis in this field and the trends in current studies are underlined.


[Prevention of gastroduodenal lesions induced by non-steroidal anti-inflammatory agents. Economic analysis from a survey of 356 physicians]. In order to obtain information on prescribing habits concerning the prevention of gastroduodenal lesions induced by non-steroidal anti-inflammatory agents (NSAI), 356 physicians practicing in 2 French departments were asked to fill a posted questionnaire. Fifty-one percent of these doctors gave an assessable answer. Among these, 84 percent occasionally prescribe "gastric protectors" associated with NSAI's in 32 percent of the prescriptions. They use antacids (48 percent), anti-H2 products (27 percent), sucralfate (11 percent) or prostaglandins (13 percent). This represents a daily cost of additional treatment ranging from 0.87 to 2.49 francs. If fibroscopies and further consultations necessitated by the prescription of NSAI's are taken into account, then 86 to 140 percent must be added to the cost of NSAI's. The profitability of these preventive measures in terms of public health will be really estimated only when the number of severe gastroduodenal lesions effectively prevented by taking topical gastric protectors or anti-secretory agents will be known.


The pancreatic duct cell.

The pancreatic duct cell. A conference entitled "The Pancreatic Duct Cell: Physiology and Pathophysiology" was held September 26-29, 1991, at the Engineering Society Club of Baltimore. The conference was organized by a committee consisting of John Williams of the University of Michigan (Co-Chair), Daniel Longnecker of Dartmouth Medical School (Co-Chair), Barry Agent of Newcastle Upon Tyne, Raymond Frizzell of the University of Alabama at Birmingham, Sherwood Githens of the University of New Orleans, and Sarah Kalser of the NIDDK. The meeting was sponsored by the NIDDK with contributions from NCI, NIDR, ADAMHA, and the American Gastroenterological Association. About 100 investigators from the United States, England, Canada, Germany, Norway, and Israel attended the conference. The participants were based in a number of distinct disciplines including both basic and clinical sciences. While the main focus was on pancreatic ducts, comparison of salivary and bile ducts was also included.


The suppression of olfactory bulbectomy-induced muricide by antidepressants and antihistamines via histamine H1 receptor blocking. The effects of antidepressants [(+)-oxaprotiline, (-)-oxaprotiline, imipramine, maprotiline, and trazodone] and antihistamines (mepyramine, dimethindene, ketotifen, methapyrilene, and antazoline) on muricidal behaviour in olfactory bulbectomized rats were investigated. All drugs except for dimethindene, which only minimally passes across the blood-brain barrier, suppressed muricide. The drugs which have high affinity for histamine H1 receptor showed potent suppressive effect on muricide. It is suggested that the central histaminergic system is involved via H1 receptors in the expression of muricide in olfactory bulbectomized rats.


Adrenal hormones in rats before and after stress-experience: effects of ipsapirone. The present study was designed to investigate the effects of the anxiolytic 5-HT1A receptor agonist ipsapirone on the hormonal responses in rats under nonstress and stress conditions by means of repeated blood sampling through an intracardiac catheter. Ipsapirone was given in doses of 2.5, 5, 10, and 20 mg/kg (IP) under nonstress conditions in the home cages of the rats. Plasma corticosterone levels increased in a dose-dependent way in the dose range of 5 to 20 mg/kg, whereas the plasma catecholamines were only significantly increased with the highest dose of the drug. The effect of ipsapirone in control and in stressed rats was studied with the selected dose of 5 mg/kg. Conditioned fear of inescapable electric footshock (0.6 mA, AC for 3 s) given one day earlier was used as stressor. Surprisingly, ipsapirone potentiated the magnitude of the neuroendocrine responses. Rats receiving an inescapable footshock 1 day earlier showed a further elevated corticosterone response to the 5-HT1A receptor agonist ipsapirone even before exposing them to the conditioned stress situation. The present findings suggest that if an animal has no possibilities to escape or avoid a noxious event, functional hypersensitivity will develop in the serotonergic neuronal system, which is reflected in the increased responsiveness of the HPA axis to a 5-HT1A agonist challenge.


Alpha 2-adrenergic modulation of pancreatic glucagon secretion in rats. The present study was designed to clarify the mechanism of adrenergic modulation of pancreatic glucagon secretion in rats under physiological conditions by 1) epinephrine infusion alone or together with adrenergic blockers and 2) administration of adrenergic agonists. Intravenous infusion of epinephrine alone (1 microgram/kg/min, equal to 0.7 nmol/kg/min) caused a significant increase in glucagon secretion. Phentolamine (an alpha blocker) or yohimbine (an alpha 2 blocker) administration completely inhibited the increase of glucagon secretion caused by epinephrine infusion, but neither the administration of bunazosin (an alpha 1 blocker) nor beta blockers inhibited it. Infusion of clonidine (an alpha 2 agonist) caused significant increase of glucagon secretion even at a low dose of 0.5 nmol/kg/min, although infusion of neither an alpha 1 nor a beta 2 agonist caused it even at the high dose of 40.0 nmol/kg/min. It is concluded that the alpha 2 receptor mechanism plays the most important role in the adrenergic modulation of glucagon secretion in rats under physiological conditions.


Influence of the acute stress on agonist-stimulated phosphoinositide hydrolysis in the rat cerebral cortex. 1. The present study was carried out in order to elucidate the influence of the acute stress on alpha 1-adrenergic, serotonin-2 (5-HT2) and muscarinic cholinergic (M-Ach) receptors-mediated phosphoinositide (PI) hydrolysis in rat cerebral cortex slices. 2. In rat cerebral cortex slices, noradrenaline (NA), serotonin (5-HT) and carbachol stimulated [3H]inositol-monophosphate (IP1) accumulation in a concentration-dependent manner. 3. The forced swimming test (FST) for 15 min induced a significant reduction of 5-HT-stimulated [3H]IP1 accumulation, but this stress situation did not produce a significant alteration of NA- and carbachol-stimulated [3H]IP1 accumulation. 4. The FST for 15 min did not affect the density and affinity of alpha 1-adrenergic, 5-HT2 and M-Ach receptors. 5. In a mild acute stress situation, the intracellular signal transduction mediated by 5-HT was promptly inhibited as compared to the signal transduction mediated by NA or carbachol. This inhibition may be induced by an acute uncoupling of 5-HT2 receptor-mediated intracellular signal transduction.


A model of interaction between Entamoeba histolytica and Shigella flexneri. The establishment of a relationship between Entamoeba histolytica and certain bacteria may contribute to the expression and/or enhancement of the pathogenicity of this parasite. Recent experiments have shown that bacteria expressing mannose-binding lectins on their surface could attach to mannose-containing molecules on the surface of amoebae. In this study, we established a model of interaction between. E. histolytica and Shigella flexneri. Using well-characterized mutants of S. flexneri, we studied the role of type I pili expression and the invasive phenotype of S. flexneri in the interaction between amoebae and the bacteria. Type I pili expression allowed attachment and subsequent internalization of S. flexneri by amoebae, these events were not observed in isogenic strains that did not express type I pili. Invasive as well as non-invasive variants of S. flexneri expressing type I pili were slowly digested by amoebae following internalization. Morphological studies showed that the specific features of the interaction depend on the dynamics of the distribution of mannose residues on the amoebic membrane during the interaction.


CD4+ cells and CD4+ percent as risk markers for Pneumocystis carinii pneumonia (PCP): implications for primary PCP prophylaxis. The incidence of Pneumocystis carinii pneumonia (PCP) during 2 years in HIV-infected patients with less than or equal to 100 x 10(6)/l CD4+ cells, less than or equal to 200 x 10(6)/l CD4+ cells and less than 20% CD4+ cells of total T lymphocytes were compared. The relative PCP risk in 57 patients with less than or equal to 100 CD4+ cells was more than twice higher than in 120 patients with less than or equal to 200 CD4+ cells. The latter had almost twice higher relative PCP risk than 271 patients with less than or equal to 20% CD4+ cells. Only 3/56 patients who acquired PCP had greater than 200 CD4+ cells and 15/56 patients had greater than 100 CD4+ cells. Centers for Disease Control (CDC) recommends primary PCP prophylaxis in HIV-infected patients when the number of CD4+ cells is less than 200 x 10(6)/l or when the CD4+ is less than 20. On the basis of the presented data we suggest that primary prophylaxis is considered only when CD4+ cells fall below 200 x 10(6)/l.


A comparison of dilution adjustment methods for urinary enzymes. Urinary dilution adjustment methods can be used to reduce the intra-individual variability in concentrations of metals and other substances in urine due to variability in urinary flow. In this study linear and non-linear dilution adjustments with urinary flow, creatinine (CREAT) and urinary density (UD) were compared for the urinary enzymes alanine amino peptidase (AAP), beta-galactosidase (beta GAL) and N-acetyl-beta, D-glucosaminidase (NAG). The most optimal dilution adjustment for AAP was: AAPadjusted = AAPmeasured/(CREATmeasured)0.824 The optimal dilution adjustment for beta GAL was: beta GALadjusted = beta GALmeasured/(CREATmeasured)0.878 For NAG the optimal dilution adjustment parameter was the conventional linear adjustment with SG. It could not be determined whether urinary dilution methods can be useful for population based reference intervals of urinary enzymes. If personal reference intervals can be calculated, urinary dilution adjustment methods may be useful by reduction of intraindividual variability.


A Mac-1 antibody reduces liver and lung injury but not neutrophil sequestration after intestinal ischemia-reperfusion. Intestinal ischemia and reperfusion (I/R) result in leukosequestration and injury to the liver and lungs. The adherence-dependent oxidative burst of neutrophils requires cell adhesion through the Mac-1 integrin. Neutrophil-mediated tissue injury may depend on this specific cell adhesion event. This study tests the effect of a Mac-1 (CD 11b) monoclonal antibody (MAb) (R17) on liver and lung injury after intestinal I/R. After collaterals were ligated in anesthetized rats, the superior mesenteric artery was clamped for 60 minutes followed by 3 hours of reperfusion. Animals were treated with saline solution, R17, or nonspecific immunoglobulin M. Another nonischemic group of rats were sham controls. Lung and intestinal polymorphonuclear leukocyte sequestration was assessed by measurement of myeloperoxidase and lung permeability by bronchoalveolar lavage blood concentration ratio of 125I-labeled bovine serum albumin. At 3 hours of reperfusion lung and intestinal myeloperoxidase and lung permeability were increased. Treatment with R17 MAb did not reduce intestinal or lung myeloperoxidase but prevented increased lung permeability. Similarly, after treatment with saline solution, liver polymorphonuclear leukocyte sequestration increased after 3 hours of reperfusion, and serum alanine aminotransferase level rose eightfold. R17 MAb did not significantly reduce liver neutrophil sequestration; however, it reduced alanine aminotransferase level more than 50% when compared to saline solution controls. At 3 hours of reperfusion there was a leukocytosis (white blood cell count, 14.9 +/- 1.0 x 10(3)/mm3 vs 6.0 +/- 0.8 in sham [p less than 0.05]). The white blood cell count was unaffected by R17 MAb. These data indicate that a MAb to the neutrophil Mac-1 integrin reduces lung and liver injury after intestinal I/R but does not reduce lung or intestinal leukosequestration.


Amplification of the HER-2/neu protooncogene in human endocrine tumors. Amplification of HER-2/neu, a protooncogene related to the epidermal growth factor receptor, has prognostic significance in patients with breast cancer. Alterations in protooncogenes have not been determined for endocrine tumors in which prognosis is difficult to predict. We have addressed the question of whether amplification of HER-2/neu or epidermal growth factor receptor occurs in DNA from human endocrine tumor lines and have sought to characterize the HER-2/neu gene and its products in carcinoid tumors of the gut. The differential polymerase chain reaction procedure was used to detect genomic amplification in DNA samples from human endocrine tumor cell lines (BON, SIM, STAN) and from paraffin-embedded samples of carcinoid tumors. Sequencing techniques were used to determine whether mutations of the transmembrane domain of HER-2/neu existed. We then further characterized the gene products (RNA and protein) in carcinoid tumors. Amplification of HER-2/neu was identified in all three endocrine tumor cell lines. HER-2/neu amplification was found in four of 10 carcinoid tumors of the gut; three of these four tumors were invasive or metastatic. In addition, HER-2/neu mRNA and protein were expressed in carcinoid tumors. Amplification of the HER-2/neu protooncogene occurs in endocrine tumors of the gut; quantitation of the actual copy number may be an important prognostic determinant. The unique human endocrine cell lines, established in our laboratory, will be useful models to further examine the significance of alterations of the HER-2/neu gene in endocrine tumors.


Presymptomatic identification of carriers of the multiple endocrine neoplasia type 2A gene using flanking DNA markers. Because the predisposition locus for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to chromosome 10 by genetic linkage analysis, it has become possible to identify gene carriers by following the transmission of linked genetic markers from affected parents to offspring at risk for MEN2A. We have applied a highly accurate genetic test to presymptomatic diagnosis of gene carriers in several large kindreds with MEN2A. DNA was extracted from 300 individuals in six kindreds with MEN2A and used for genotyping studies with DNA markers flanking the MEN2A locus. Genotype data were used to predict the inheritance of the MEN2A gene in kindred members at risk according to previously calculated map distances and the program LINKAGE: Ninety-five percent of individuals were informative with markers flanking the MEN2A locus. Of 130 patients at risk, 26 (20%) were predicted to be MEN2A gene carriers, 100% (77%) were noncarriers, and 4 (3%) were recombinant and their gene carrier status could not be determined. Gene carrier prediction probabilities were calculated at greater than 98% in 94% of these patients. We conclude that genetic testing with flanking DNA markers is a highly accurate method for the presymptomatic identification of MEN2A gene carriers and allows for diagnosis at an earlier stage than does traditional calcitonin testing.


Decreased leukocyte adhesion with anti-CD18 monoclonal antibodies is mediated by receptor internalization. Adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells is mediated partially by CD11/CD18 integrins. The purpose of this study was to define (1) the response of PMNs to anti-CD18 monoclonal antibody binding, and (2) the mechanism responsible for anti-CD18 monoclonal antibody-mediated decreases in PMN adhesion to endothelial cells. Canine PMN O2- production, myeloperoxidase, and lysozyme release in response to the anti-CD18 monoclonal antibody IB4 were measured by standard assays. To examine endocytosis of CD18 receptors, PMNs incubated with IB4 and a fluorescein isothiocyanate secondary antibody were analyzed by flow cytometry. Treatment of PMNs with IB4 did not stimulate O2- production or degranulation but decreased adhesion of 51Cr-labeled PMNs to ex vivo canine aorta. Incubation of PMNs at 25 degrees C resulted in a decrease in fluorescence intensity that was not affected by NaN3 or vanadate but was blocked by NaF, 4 degrees C, and bafilomycin, which prevents endosomal acidification. Treatment with an antifluorescein antibody decreased the fluorescence intensity in NaF and 4 degrees C, but not in bafilomycin-treated neutrophils. IB4 decreases PMN-endothelial cell adhesion but does not stimulate neutrophil oxidative metabolism or degranulation. These data suggest that reduced adhesion may be the result of internalization of the CD18/IB4 complex. Anti-CD18 monoclonal antibodies may be useful in preventing PMN adhesion without the potentially deleterious effects of cell activation.


Independent modulation of enterocyte migration and proliferation by growth factors, matrix proteins, and pharmacologic agents in an in vitro model of mucosal healing. Gastrointestinal mucosa heals by restitution and proliferation. These are difficult to distinguish in vivo. Human Caco-2 enterocytes were cultured on matrix proteins (collagen I, laminin, fibronectin) with growth factors (epidermal growth factor [EGF] and transforming growth factor-beta 1 [TGF-beta 1]) and the tyrosine kinase and prostaglandin inhibitors genistein and indomethacin. Healing was modeled by means of monolayer expansion, proliferation by means of 3H-thymidine uptake, and restitution by means of mitomycin-blocked migration. Changing matrix composition failed to alter proliferation, but collagen I stimulated migration more than laminin or fibronectin (laminin/collagen, 68% +/- 2%; p less than 0.05). EGF (30 ng/ml) increased proliferation on both collagen (225% +/- 11% of basal) and laminin (206% +/- 26%) but increased migration only over laminin (210% +/- 17%) (all, p less than 0.05). TGF-beta 1 (200 pg/ml) stimulated migration over laminin (187% +/- 18%, p less than 0.005) but inhibited migration over collagen (89% +/- 3%, p less than 0.01) and did not affect 3H-thymidine uptake. When cultured on laminin, EGF but not TGF-beta 1 altered organization of the alpha 2 integrin subunit. Genistein (100 mumol/L) inhibited basal and EGF-stimulated 3H-thymidine uptake. In addition, it prevented EGF stimulation of replication-blocked migration (81% +/- 10% vs 190% +/- 20% of basal, p less than 0.0001) without altering basal replication-blocked migration. Indomethacin (10(-5) mol/L) did not alter migration but inhibited basal and EGF-stimulated proliferation by 7% +/- 1% (each, p less than 0.005). Restitution and proliferation appear independently regulated by matrix and growth factors. It may be possible to individually target specific phases of mucosal healing by means of pharmacologic agents.


Immune status of asymptomatic HIV-infected hemophiliacs: randomized, prospective, two-year comparison of treatment with a high-purity or an intermediate-purity factor VIII concentrate. It has been postulated that high-purity factor VIII (FVIII) concentrates, since they contain less alloantigenic proteins than intermediate-purity concentrates, might cause lesser deterioration of the immune systems of hemophilic patients infected with the human immunodeficiency virus (HIV). To evaluate this hypothesis, we have prospectively compared T-lymphocytes subsets and delayed hypersensitivity reactions to skin tests in 17 asymptomatic HIV-positive hemophiliacs randomly assigned to continue treatment with an intermediate-purity concentrate with those of 16 hemophiliacs changed to a high-purity concentrate. For both groups, during the 24-month follow-up period CD4 cell counts showed similar rates of fall from baseline values. There was also no difference in the number of patients anergic to skin tests. Three patients treated with the intermediate purity concentrate and one treated with the high-purity concentrate developed symptoms of HIV infection. On the whole, no striking benefit is conferred to the immune status of asymptomatic HIV-positive hemophiliacs by using this high-purity concentrate for 2 years.


Activation of bovine plasma benzylamine oxidase (BzAO) by 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine. We have shown previously that certain analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are potent inhibitors of human and bovine plasma benzylamine oxidase (BzAO: EC 1.4.3.6). Inhibition was competitive, reversible and allosteric. Under certain conditions competitive inhibitors of allosteric enzymes can act as allosteric activators. In the present work, 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3MPTP) was found to activate bovine plasma BzAO at low substrate and 2'-CH3MPTP concentrations. At higher 2'-CH3MPTP concentrations, the activation was negated.


[The role of intermediate filaments in forming cellular processes induced in fibroblasts by the tumor promoter TPA]. A tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes in the cell shape in certain fibroblastic lines. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Two specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. A short (I h) exposure to PMA induced formation of processes in the control cells rather than in the Colcemid treated cells having depolymerized microtubules and the IF that collapsed around the nucleus. A longer (3-4 h) exposure to PMA of the colcemid-treated cells induced a partial reversal of the IF collapse; those parts of peripheral lamellae that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of the IF with the actin system is an essential step in the formation of processes from lamellae.


Distribution of polymorphic HLA-DR and -DQ alleles as determined by restriction fragment length polymorphism analysis in an Austrian population. The restriction fragment length polymorphism (RFLP) of HLA-DR beta, -DQ alpha and -DQ beta genes was analyzed in 637 unrelated individuals from the Austrian population. The restriction enzyme was Taq I, and three exon-specific probes were applied. The gene frequencies, the Hardy-Weinberg equilibrium in DR beta and DQ loci, linkage disequilibria between the loci and haplotype frequencies are calculated. Rare associations between DR and DQ loci are described. Two RFLP patterns are demonstrated which were unique in the overall 1,000 individuals tested so far.


Combination of GM-CSF and cytosine in myelodysplasia results in improved neutrophil function. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given concurrently with low-dose cytosine arabinoside for 3 weeks to patients with myelodysplasia. Neutrophil activation as evidenced by increased chemiluminescence and reduced surface expression of CD16 was consistently seen during therapy. An attendant fall in chemotaxis was also observed. These effects occurred even when neutrophil counts did not rise significantly at lower doses of GM-CSF. Although no improvement in anaemia or thrombocytopenia was observed, the neutrophil counts became normal during therapy without significant expansion of marrow cellularity or colony-forming ability. No major toxicities were observed, even at higher dosages of GM-CSF.


Immunohistochemical localization of calcitonin gene-related peptide and cotransmitters in a subpopulation of post-ganglionic neurons in the porcine inferior mesenteric ganglion. Applying double-fluorescence immunohistochemistry, adrenergic and non-adrenergic postganglionic sympathetic neurons, in the porcine inferior mesenteric ganglion (IMG) are subdivided according to size and cotransmitter content. Calcitonin gene related peptide (CGRP)-immunoreactive (IR) neurons are demonstrated to belong to the non-adrenergic, i.e. tyrosine hydroxylase- and DOPAmine-beta-hydroxylase-(D beta H)-negative subpopulation of postganglionic perikarya. Virtually all of the CGRP-IR postganglionic neurons exhibit colocalization with somatostatin (SOM), and, some of them with neuropeptide tyrosine (NPY). Additionally, NPY-, SOM-, and NPY/SOM-IR subpopulations of adrenergic and non-adrenergic neurons are observed. CGRP-immunoreactivity is seen in dense networks of intraganglionic varicose nerve fibres, adjacent to the TH- and SOM-IR neurons. NPY-IR perikarya are sparsely supplied by CGRP-IR fibres. SOM- and NPY-IR nerve fibres also exist in the inferior mesenteric ganglion. The functional relevance of CGRP-IR postganglionic neurons, as well as target organs of these neurons remain to be elucidated.


Glutamate and GABA levels in CSF from patients affected by dementia and olivo-ponto-cerebellar atrophy. The modifications in the CSF content of glutamate and GABA in patients afflicted with primary degenerative dementia (PDD) and olivo-ponto-cerebellar atrophy (OPCA) have been evaluated. Control subjects (with disk herniation) were also included in the study. The amino-acids assays were carried out utilizing enzymatic-bioluminescence technique. GABA levels in controls were 803 +/- 98 (n = 7) and in demented patients 702 +/- 98 (n = 7) pmol/ml. Glutamate levels were 2067 +/- 244 (n = 10) in controls, 1190 +/- 81 (n = 16) pmol/ml (vs controls p less than 0.01) in demented patients, and 1116 +/- 146 (vs controls p less than 0.01) in OPCA patients. These results suggest that CSF glutamate levels in severely demented patients might be a result of generalized neuronal loss in the brain with a reactive gliosis.


Common factors contributing to intractable pain and medical problems with insufficient drug uptake in areas to be treated, and their pathogenesis and treatment: Part I. Combined use of medication with acupuncture, (+) Qi gong energy-stored material, soft laser or electrical stimulation. Most frequently encountered causes of intractable pain and intractable medical problems, including headache, post-herpetic neuralgia, tinnitus with hearing difficulty, brachial essential hypertension, cephalic hypertension and hypotension, arrhythmia, stroke, osteo-arthritis, Minamata disease, Alzheimer's disease and neuromuscular problems, such as Amyotrophic Lateral Sclerosis, and cancer are often found to be due to co-existence of 1) viral or bacterial infection, 2) localized microcirculatory disturbances, 3) localized deposits of heavy metals, such as lead or mercury, in affected areas of the body, 4) with or without additional harmful environmental electro-magnetic or electric fields from household electrical devices in close vicinity, which create microcirculatory disturbances and reduced acetylcholine. The main reason why medications known to be effective prove ineffective with intractable medical problems, the authors found, is that even effective medications often cannot reach these affected areas in sufficient therapeutic doses, even though the medications can reach the normal parts of the body and result in side effects when doses are excessive. These conditions are often difficult to treat or may be considered incurable in both Western and Oriental medicine. As solutions to these problems, the authors found some of the following methods can improve circulation and selectively enhance drug uptake: 1) Acupuncture, 2) Low pulse repetition rate electrical stimulation (1-2 pulses/second), 3) (+) Qi Gong energy, 4) Soft lasers using Ga-As diode laser or He-Ne gas laser, 5) Certain electro-magnetic fields or rapidly changing or moving electric or magnetic fields, 6) Heat or moxibustion, 7) Individually selected Calcium Channel Blockers, 8) Individually selected Oriental herb medicines known to reduce or eliminate circulatory disturbances. Each method has advantages and limitations and therefore the individually optimal method has to be selected. Applications of (+) Qi Gong energy stored paper or cloth every 4 hours, along with effective medications, were often found to be effective, as Qigongnized materials can often be used repeatedly, as long as they are not exposed to rapidly changing electric, magnetic or electro-magnetic fields. Application of (+) Qi Gong energy-stored paper or cloth, soft laser or changing electric field for 30-60 seconds on the area above the medulla oblongata, vertebral arteries or endocrine representation area at the tail of pancreas reduced or eliminated microcirculatory disturbances and enhanced drug uptake.(ABSTRACT TRUNCATED AT 400 WORDS)


Clinical research on treating senile dementia by combining acupuncture with acupoint-injection. Combining acupuncture with acupoint-injection of aceglutamidi has been used in treating 38 cases of senile dementia. Our experiment showed that the therapy is effective for the cases of multi-infarct dementia, the rate of success being 42.85% and of improvement 42.86%, the total efficacy rate being 85.71%. The rating was based on the revised Hasegawa Dementia Scale and the Functional Activity Questionnaire. In addition, it has been observed that the component of high density lipid-cholesterone increased significantly after treatment.


Detection of extraordinary large bio-magnetic field strength from human hand during external Qi emission. It is generally accepted that more than 10(-6) gauss order magnetism was not detected in normal human condition. However, we detected 10(-3) gauss (mGauss) order bio-magnetic field strength from the palm in special persons who emitted External Qi ("Chi" or "Ki"). This detection was possible by special arranged magnetic field detection system, consisted of a pair of 2 identical coils with 80,000 turns and a high sensitivity amplifier. Each of the coils were rolled 80,000 turns accurately, and were connected in series in opposite direction, actuating as a gradiometer. We measure bio-magnetic field strength in 37 subjects with this detection system. The only 3 subjects of them exhibited strong bio-magnetic field of 2 to 4 mGauss in frequency range of 4 to 10 Hz. This magnetic field strength was greater than that of normal human bio-magnetism by 1,000 times at least. A simultaneous measurement of bio-magnetic field strength and its corresponding bio-electric current was examined in one subject. During exhibiting such strong bio-magnetism, its corresponding electric current was not detectable. Therefore, the extra-ordinary large bio-magnetic field strength can not derive from internal body current alone, hence the origin of the large bio-magnetism is still unknown. We suppose that the extraordinary large bio-magnetic field strength might be originated from "Qi" energy in the oriental medicine or in the oriental traditional philosophy.


Acupuncture-like stimulation with codetron for rehabilitation of patients with chronic pain syndrome and osteoarthritis. Acupuncture is one of the oldest healing methods which is used in traditional medicine. In the modern medicine, we are witnessing a renaissance of this ancient treatment applied mainly in the management of chronic pain. A number of modern technological changes are being applied to replace, or modify, the classical needle treatment. Among many modalities used today is the novel addition in Transcutaneous Electrical Nerve Stimulation (TENS) called CODETRON which delivers acupuncture-like stimulation in a random order. CODETRON was developed by a Canadian Scientist and had been evaluated in a clinical trial in a multidisciplinary pain clinic on patients who came for acupuncture therapy over a period of two years. Indications, effectiveness and experiences with this form of treatment are presented. In addition, results obtained from a six week double-blind randomized placebo controlled pilot trial of osteoarthritis of the hip/knee with CODETRON which was conducted later. The results were highly suggestive of the beneficial effect of this nonhabituating mode of therapy and confirmed our initial uncontrolled trial results.


Treating poisonings: focus on syrup of ipecac. The awareness of poison prevention is increasing despite an alarming incidence of accidental and intentional poisonings in the United States. During 1990, 72 poison centers throughout the United States, serving a population of 191.7 million, submitted to the American Association of Poison Control Centers data collection system 1,713,462 cases of poisonings, which included 612 deaths. Emetic agents, when used properly, can reduce the extent and severity of improperly ingested medications, selected household products, and other toxic chemicals. The pharmacist can play a valuable role in distributing information about poison control centers, poison prevention, and appropriate treatment of poisonings.


Sulfasalazine desensitization in children and adolescents with chronic inflammatory bowel disease. Sulfasalazine is an important therapeutic agent in the management of chronic inflammatory bowel disease (CIBD). Unfortunately, adverse reactions to this drug have been reported in 5-55% of treated patients. These include dose-related side effects like nausea, malaise, and headache or hypersensitivity reactions such as rash, fever, hives, arthralgia, hepatitis, etc. Studies in adults with successful reintroduction of sulfasalazine after a desensitization program have been reported; however, with regard to children, no such data are available. Fourteen children and adolescents (5-16 yr old) diagnosed to have CIBD manifested hypersensitivity to sulfasalazine within 2 months of onset of treatment. All had pancolitis--secondary to Crohn's disease (CD) in four and to ulcerative colitis (UC) in 10. All of them were on steroids. Sulfasalazine was discontinued in all after symptoms of hypersensitivity developed. Three patients with severe reaction were diagnosed prior to desensitization experience. Desensitization, beginning with 5-50 mg of sulfasalazine/day, was attempted in the other 11 children. The dose was gradually increased by 5-50 mg increments every 3 days. Desensitization was successful in only five children, who were ultimately able to tolerate 1.5-3.0 g of sulfasalazine daily again. In the rest (six of 11 patients), oral 5-ASA (Asacol) was administered, and three could not tolerate it. One of these three with intolerance to Asacol required colectomy. One did not tolerate Asacol or Dipentum. Our findings suggest that sulfasalazine desensitization should be attempted in all patients developing hypersensitivity reactions before trying alternative therapy.


Physician assistants in gastroenterology: should they perform endoscopy? New technology and increased screening examinations continue to cause the demand for gastrointestinal endoscopic procedures to mushroom. Hospitals administered by the Department of Veterans Affairs, public health care facilities, and health maintenance organizations struggle to meet this demand because of limited resources for expansion of space or staff. For the past 3 yr, we have used a physician assistant in our endoscopy laboratory to assist with and perform endoscopic procedures. The benefits for our hospital include increased staff efficiency, improved housestaff education, and ability to perform an increased number of procedures with existing staff.


Optimizing the information obtained from continuous 24-hour gastric pH monitoring. Our objective in performing this study was to determine the best way to summarize experimental data obtained from 24-h continuous intragastric pH-metry. For this purpose, we reviewed 605 circadian intragastric pH recordings performed under different clinical conditions. Included were 82 normal subjects, 126 patients with active duodenal ulcer, 272 ulcer patients in clinical remission treated with a single bedtime dose (at 10 PM) of various H2 antagonists and 125 patients with active ulcer treated with a single morning dose (8 AM) of omeprazole. For each group, we calculated the time spent at different pH levels scaled with 0.1 incremental steps in three different time intervals: 24 h (5 PM to 4:59 PM), daytime (8 AM to 7:59 PM), and nighttime (8 PM to 7:59 AM). Then we calculated the mean and the median pH, as well as the time spent in min greater than 3.0 pH units over a mainly drug-related time window (8 PM to 7:59 AM) for each treatment and its respective placebo group. The mean and the median pH, as well as the time spent in minutes greater than 1.2 pH units over 24 h were also calculated for the untreated groups of normal subjects and duodenal ulcer patients. Considering the individual pH recordings, bi- or plurimodal density distributions of pHs were found in more than 90% of the 605 subjects, no matter the time window and the clinical condition examined, and log-normal distributions were found in only three cases. Considering the groups instead of single pH recordings, conversely, normal subjects and duodenal ulcer patients showed average left-skewed unimodal density distributions, whereas bimodal patterns were the rule in the groups of patients treated with H2 antagonists and omeprazole. The time spent greater than 3.0 pH units was the most powerful acidity index in assessing drug effectiveness in the various subsets of antisecretory compounds, in that the Student's t test values obtained with this variable were almost invariably higher than those obtained with the mean and median pH levels. Also, the time spent greater than 1.2 pH units was the most powerful summary variable in differentiating normal subjects from duodenal ulcer patients (p less than 0.01, compared with the mean and median pH). We conclude that the time spent above a given pH threshold of interest is a more accurate index than the mean and median pH in summarizing gastric acidity changes under all clinical conditions, and this is because it better represents the experimental pH measurements from which it comes.


Diagnostic value of serum gamma-glutamyl transferase isoenzyme for hepatocellular carcinoma: a 10-year study. Serum gamma-glutamyl transferase (GGT) was separated into nine to 11 isoenzyme bands (designated as GGT I-XI) by vertical slab electrophoresis on polyacrylamide gradient gel. The diagnostic value of GGT isoenzyme II (GGT II) for hepatocellular carcinoma (HCC) was studied, and the results were as follows: 1) GGT II was positive in 90% of 90 cases of HCC, and negative in most patients with acute and chronic viral hepatitis, extrahepatic tumors, in pregnant women, and in healthy controls; 2) the positive rate of GGT II assay was higher than that of alkaline phosphatase isoenzyme I (ALP I), alpha-fetoprotein (AFP), and alpha 1-antitrypsin (AAT) in 101 cases of HCC. In cases in which the AFP was greater than 50 ng/ml or less than 50 ng/ml, the positive rates of GGT II were 70.8% and 75-100%, respectively; 3) of 14 cases of small-size HCC, the positive rate of GGT II was 78.6%, which was higher than that of AFP (50%), AAT (28.6%), and ALP I (0%); 4) of 62 cases that were false-positive for GGT II assay, 24.2% developed into HCC during a follow-up of 2.1-20 months. In subjects with persistent and recurrent positivity of GGT II, 86.7% and 22.2%, respectively, developed HCC. No patient with temporal positivity of GGT II developed HCC. The results show that GGT II can be applied as an additional marker for HCC, and is valuable not only for the diagnosis of clinical HCC, but for the detection of small or subclinical HCC. Periodic follow-up with assay of GGT II in patients at high risk for HCC may predict the development of hepatoma.


Expected behavior of conditional linkage disequilibrium. The ubiquitousness of RFLPs in the human genome has greatly helped the mapping of human disease genes, and it has been suggested that population measures of association between disease and marker loci could help with this mapping. For rare diseases, random samples are taken from within disease genotypes in order to obtain reasonable sample sizes, but this sampling strategy requires a modification of the usual measures of association. We present theoretical predictions for the mean and variance of such a modified measure, under the assumption that the disease gene is maintained at a constant low frequency in the population. The coefficient of variation of this modified measure is large enough that caution is needed in using the measure to locate disease genes, and, furthermore, the coefficient of variation cannot be made arbitrarily small by increasing sample size. The modified association measure is calculated for recently published data on cystic fibrosis.


Molecular abnormalities of a human glucose-6-phosphate dehydrogenase variant associated with undetectable enzyme activity and immunologically cross-reacting material. Among a large number of glucose-6-phosphate dehydrogenase (G6PD) variants associated with different severity of clinical manifestations, enzyme deficiency, and kinetic abnormalities found in humans, only one variant exhibits no measurable activity and lacks an immunologically cross-reacting material in blood cells and other tissues. The mRNA content of the patient's lymphoblastoid cells was found to be normal, and the size of mRNA was also normal (i.e., approximately 2.4 kb). Western blot hybridization indicated that the patient's cells did not produce cross-reacting material. The variant mRNA was reverse transcribed and amplified by PCR. Nucleotide sequencing of the variant cDNA showed the existence of three nucleotide base changes, i.e., a C----G at nucleotide 317 (counting from adenine of the initiation codon), which should cause Ser----Cys substitution at the 106th position (counting from the initiation Met); a C----T at nucleotide 544, which induces the Arg----Trp at the 182d position; and a C----T at nucleotide 592, which induces Arg----Cys at the 198th position of the protein. The existence of three mutation sites was confirmed by sequencing of selected regions of the variant gene. No base deletion or frameshift mutation was found in the variant cDNA. No nucleotide change was detected in the extended 5' region, which included the most distal cap site. When the variant cDNA was expressed in Escherichia coli, the G6PD activity was approximately 2% of that expressed by the normal cDNA, and cross-reacting material was undetectable. However, when the variant mRNA was expressed in the in vitro translation system of rabbit reticulocytes, the variant protein was produced. These results suggest that extremely rapid in vivo degradation or precipitation of the variant enzyme induced by the three amino acid substitutions could be the major cause of the molecular deficiency.


Spondyloepiphyseal dysplasia in a Cape Town family: linkage with the gene for type II collagen (COL2A1). A moderately severe form of autosomal dominant (AD) spondyloepiphyseal dysplasia (SED) has been documented in 14 individuals in 3 generations of a family in Cape Town, South Africa. Affected persons had a short trunk; radiographic investigations indicated that skeletal involvement was worst in the hips and spine. Linkage studies with restriction fragment length polymorphisms (RFLPs) associated with the COL2A1 gene and the phenotype yielded a maximal LOD score of 4.51 at theta = 0.00. This result suggests that the structural locus for type II collagen is primarily involved in the pathogenesis of this form of SED.


Cytogenetic, biochemical, and molecular analyses of a 22q13 deletion. We report on a 3-year-old boy with a terminal deletion of 22q. The activity of alpha-N-acetylgalactosaminidase was normal while arylsulfatase A activity was reduced. Molecular analysis demonstrated the lack of paternal alleles of D22S45 and D22S55.


Iontophoretic study of speed of action of various muscle relaxants. The speed of action of nondepolarizing muscle relaxants is inversely related to potency. The hypothesis that this effect occurs at the end plate was tested in a frog (Rana pipiens) cutaneous pectoris muscle preparation. Brief acetylcholine pulses (10-100 ms) were applied iontophoretically from a central barrel of a triple-barrelled microelectrode located near an end plate. Long pulses (10-200 s) of muscle relaxant (gallamine, rocuronium, d-tubocurarine, atracurium, vecuronium, pancuronium, and doxacurium) were applied from one of two other barrels. The responses were a voltage change at the end plate, measured with an intracellular electrode. To evaluate potency, intracellular voltage changes following iontophoretic acetylcholine pulses were measured after application of various concentrations of muscle relaxants. The following were the equilibrium dissociation constants, which represent concentration of relaxant for 50% inhibition of response (mean plus or minus standard deviation): gallamine, 4.56 +/- 0.44 microM (n = 5); rocuronium, 0.71 +/- 0.09 microM (n = 6); d-tubocurarine, 0.59 +/- 0.07 microM (n = 4); atracurium, 0.31 +/- 0.03 microM (n = 4); vecuronium, 0.23 +/- 0.02 microM (n = 5); pancuronium, 0.18 +/- 0.03 microM (n = 3); doxacurium, 0.11 +/- 0.03 microM (n = 5). Both onset and offset of effect of muscle relaxant proceeded with an exponential time course.(ABSTRACT TRUNCATED AT 250 WORDS)


Potential histamine H2-receptor antagonists: synthesis and pharmacological activity of derivatives containing acylamino-furazan moieties. Analogues of 3-amino-4-[2-[(5-dimethylaminomethyl-2-furyl)methylthio]ethylamino] furazan (1) containing carbonyl groups joined to the amino functions linked to the furazan system have been synthetized and investigated for their H2-antagonist properties on the isolated guinea pig right atrium. The presence of the carbonyl group lowers the activity in respect to the corresponding leads. The decrease in activity is only by 1-2 orders of magnitude in the 3-acylamino-furazan series versus inactivity in the 4-acylamino isomers and in the diacylated series.


Complications following pancreaticoduodenectomy. Current management. From 1980 to 1989, 279 patients underwent pancreaticoduodenectomy at a single institution with a postoperative mortality of 4%. The aim of this study was to determine incidence, origin, and present management strategy of early complications following this operation. Significant morbidity occurred in 46% of the patients, including delayed gastric emptying (23%), pancreaticojejunal anastomotic leak (17%), intra-abdominal sepsis (10%), biliary-enteric anastomotic leak (9%), gastrointestinal tract bleeding (5%), and intra-abdominal hemorrhage (3%). Complications were associated with advanced age, prolonged operations, and increased operative blood loss. Most complications were managed nonoperatively. Mortality was increased when a reoperation was required, a biliary-enteric leak occurred, or an intra-abdominal abscess developed. Pancreaticoduodenectomy continues to carry a high postoperative morbidity; however, operative mortality is low, and management of complications has been made simpler with more sophisticated, nonoperative therapeutic options.


Drug-induced movement disorders in institutionalised adults with mental retardation: clinical characteristics and risk factors. Fifty-three institutionalised adults with mental retardation, the majority (73.5%) moderate to severe, were examined for drug-induced movement disorders. Using a global AIMS score of 2 or more, 16 (34%) of the 47 subjects who had been exposed to neuroleptics had tardive dyskinesia (TD). Three of these had developed the dyskinesia upon withdrawal of neuroleptics. The dyskinetic movements were mainly seen in the lingual, perioral and other facial muscles. Two (33%) out of 6 subjects with no history of exposure to neuroleptics also had similar dyskinetic movements. The total neuroleptic dose significantly, and age marginally, but not sex, brain damage or level of mental retardation, emerged as risk factors for TD. Two (3.7%) subjects had definite akathisia and 16 (30.8%) significant extrapyramidal side effects. This study supports the findings of previous studies of considerable neurological adverse effects of neuroleptics in this patient group and cautions against their injudicious use. It provides further evidence for some putative risk factors for TD and is noteworthy for its lack of support for the contentious issue of brain damage as a risk factor.


Neuroleptic prescription for Chinese schizophrenics in Hong Kong. There are very few studies on the pattern of neuroleptic prescription for schizophrenics in Asia. 106 schizophrenic patients in a psychiatric unit of a general teaching hospital in Hong Kong were surveyed. The mean daily dose (in chlorpromazine equivalent) was low (568.5mg). The mean daily dose of high potency agents was four times that of low potency agents. A high frequency of use of anticholinergic drugs may indicate that Chinese are more susceptible to acute extrapyramidal side-effects.


Beta-blockers: propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Elimination of propranolol atherogenic effects by papaverine. The addition of the beta-blockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol to a culture of peritoneal macrophages or smooth muscle cells induced an increase in the intracellular cholesterol content. Blood serum obtained from a rabbit after a peroral administration of beta-blockers also induced cholesterol accumulation. This property of drug or blood serum obtained after peroral administration is conventionally referred to as atherogenic potential or atherogenicity. Regular administration of propranolol during a 21-day period evoked stable atherogenicity of rabbit blood serum. This was accompanied by stimulation of manifestations of atherosclerosis in the aorta deendothelialized with a balloon catheter. Propranolol increased neointimal thickening, lipid accumulation, an increase in cell number and in the collagen content. In vitro, the combination of propranolol with papaverine eliminated the atherogenic effect of propranolol which manifested itself as stimulation of cholesterol accumulation in cultured cells. Simultaneous peroral administration of propranolol and papaverine prevented the appearance of serum atherogenicity. Papaverine eliminated neointimal thickening, an increase in cell number and in the lipid and collagen contents evoked by propranolol. Papaverine itself had no effect on these parameters. Thus, the atherogenicity of propranolol as well as capacity of papaverine to eliminate beta-blocker atherogenicity revealed in cell culture was confirmed in vivo. We hope that these results may be useful in the development of new drugs and optimization of antiatherosclerotic drug therapy.


Long QT syndrome presenting as a seizure. A 21-year-old woman was brought to the emergency department after being found unconscious in a hotel lobby. On presentation, she was awake but confused. The initial evaluation revealed no evidence of trauma, metabolic abnormality, drug ingestion, or intracranial process. The only abnormality noted was electrocardiographic, and included a long QT interval as well as occasional atrial and junctional beats within a normal sinus rhythm. While in the department the patient developed tonic-clonic activity and was concurrently noted to have developed ventricular tachycardia. A precordial thump was given with the simultaneous cessation of the arrythmia and the seizure. After definitive electrophysiologic study, the diagnosis of long QT syndrome was made. Treatment consisting of beta blockade and pacemaker insertion prevented further arrythmia or seizure activity. Long QT syndrome should be considered a possible etiology in any patient presenting with new onset seizures, especially in the young.


High pressure industrial steam washer injury resulting in digital amputation. High pressure washers are increasingly used to clean industrial machinery. We report a case of severe full skin thickness burn resulting from momentary exposure to the jet of steam.


Does a subparalysing dose of vecuronium enhance diaphragm fatigue? We have examined, in six healthy volunteers, the effect of a subparalysing dose of vecuronium on the development of diaphragm fatigue. Vecuronium was given as a 0.5-mg bolus i.v. followed by 0.5 mg infused over 30 min; as a control, saline was given in random order. Diaphragm strength was assessed by measuring transdiaphragm pressure and by electromyography. Diaphragm fatigue was induced by breathing against an inspiratory resistance. The plasma concentration of vecuronium varied between 15 and 30 ng ml-1 15 min after administration of vecuronium was started. Peripheral neuromuscular block was not detected in any subject. Diaphragm fatigue developed within the same period in both groups: mean 334 (SD 166) s after saline and 345 (190) s after vecuronium. The electromyographic pattern of diaphragm fatigue and the time constant of relaxation of transdiaphragm pressure after fatigue were similar in both groups. We conclude that, at low plasma concentrations of vecuronium, similar to those present in the postoperative period, there was no predisposition to diaphragm fatigue.


Effect of doxapram on neostigmine evoked antagonism of vecuronium neuromuscular block. We have examined the effect of doxapram on neostigmine-evoked antagonism of vecuronium neuromuscular block in a double-blind study in anaesthetized patients. Neuromuscular transmission was measured with a Datex Relaxograph. The mean time to recovery of the first contraction of the train-of-four (T1) from 25% to 75% was significantly longer in the presence of doxapram (138 (SEM 21) s; n = 23) than in its absence (95 (13) s; n = 29) (P = 0.014). The recovery of the train-of-four ratio was prolonged also in the presence of doxapram, although this difference was not statistically significant.


Effect of doxapram on the rate of recovery from atracurium and vecuronium neuromuscular block. We have studied the effect of doxapram on the rates of spontaneous and neostigmine-induced recovery from neuromuscular block with atracurium and vecuronium, by measurement of the time to recovery of T1 (first twitch in the train-of-four) from 25 to 75% of control (recovery index, RI). After each neuromuscular blocking drug, RI was measured without administering either doxapram or neostigmine (control group), or after administration of doxapram 1 mg kg-1, neostigmine 50 micrograms kg-1 or a combination of doxapram and neostigmine, in groups of 10 patients. RI was significantly longer after vecuronium in the presence of doxapram compared with control (20.1 min vs 14.6 min). There was no significant difference in the RI after atracurium in the presence of doxapram compared with control (12.5 min vs 11.8 min) or when neostigmine was administered with or without doxapram (2.4 min vs 2.4 min, respectively after vecuronium; 3.3 min vs 2.9 min, respectively, after atracurium).


Stereoselectivity at alpha-adrenoreceptor subtypes: observations with the enantiomers of WB 4101 separated through their amides of N-tosyl-(S)-proline. We present a chromatographic method for the separation and determination of the optical purity of the enantiomers of WB 4101 [(+/-)-1], one of the most potent and selective alpha 1-adrenoreceptor antagonists. (+/-)-1 was converted into the amide of N-tosyl-(S)-proline. The two diastereoisomers were separated on silica gel and analysed by HPLC reversed phase. The analytical method described is both accurate and sensitive and allows the optical purity to be determined at very low concentrations and to obtain WB 4101 enantiomers with a purity of more than 99.95%.


Expression of multidrug resistance gene mdr1 mRNA in a subset of normal bone marrow cells. The multidrug resistance gene mdr1, encoding P-glycoprotein (P-gp), can be expressed at high levels in tumour cells derived from normal tissues with constitutive high expression of this gene. In myelogenous leukaemia, the incidence of increased expression of mdr1 gene contrasts with the low expression of this gene in normal bone marrow (b.m.). To detect cells expressing mdr1 gene in normal and post-chemotherapy b.m., we used in situ RNA hybridization and RNA phenotyping by the polymerase chain reaction for mdr1 mRNA detection. The presence of P-gp was evaluated by immunocytochemistry with MRK16. Fifteen b.m. (eight normal and seven post chemotherapy) were tested by in situ hybridization and either PCR (three b.m.) or immunocytochemistry (11 b.m.) or both (one b.m.). With in situ mRNA hybridization, a subset (7.7% +/- 3.1%) of b.m. cells expressed mdr1 mRNA in all cases tested, with no significant differences between normal b.m. and post chemotherapy b.m. 18% of myeloid recognizable cells and 7% of the cells with lymphoid morphology expressed mdr1 mRNA. By RNA phenotyping, the four samples tested for in situ hybridization and two additional post chemotherapy b.m. expressed mdr1. MRK16 was unable to detect a significant number of cells expressing P-gp either by immunocytochemistry in the 12 b.m. tested for in situ hybridization (0% in nine cases; 0.4%, 1% and 3% of positive cells in three cases), or by flow cytometry in six additional normal b.m. (0-1.4% positive cells).


Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs. The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.15 mM) were all approx. 5 mumol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-Sn' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GFR(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, Km: Abu less than Ala less than Pro less than Val less than Ser less than Gly much less than Leu; kcat: Pro greater than Ala greater than Abu greater than Ser greater than Gly much greater than Leu greater than Val; kcat/Km: Abu greater than Pro greater than Ala much greater than Ser greater than Gly = Val much greater than Leu. Km is at a minimum and kcat/Km at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.


Retroviral gene therapy and its application in oncohematology. A symposium entitled "Foetal and Neonatal Cell Transplantation and Retroviral Gene Therapy" recently organized under the aegis of the Mérieux Foundation in Annecy, France, brought together 100 scientists and clinicians from European countries and the United States. The last day of the meeting focused on retroviral gene therapy in oncohematology. The speakers provided the basis for therapeutic applications of gene transfer and showed practical directions to be followed in the near future. Although it may be mainly restricted to in vitro manipulation of human cells at start, gene therapy is already applicable to the treatment of genetic disorders, cancer, and viral infections. The reality of gene therapy was well illustrated by the nature of the questions raised by clinicians and scientists during the meeting.


Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe. A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.


Mitochondrial DNA polymorphism in dogs.

Mitochondrial DNA polymorphism in dogs. Mitochondrial DNA (mtDNA) polymorphism was studied in 20 mongrel dogs using 14 restriction enzymes. The polymorphism was observed in the cleavage patterns of Apa I, EcoR I, EcoR V, Hinc II and Sty I. Three morphs using EcoR I and Apa I and two morphs using EcoR V, Hinc II and Sty I were found. However, no polymorphism was detected in the cleavage patterns of BamH I, Bgl II, Hae II, Hind III, Pst I, Sca I, Stu I and Xba I. The examined dogs were classified in seven types using five restriction endonucleases which recognized six nucleotide sequences. The value of nucleotide diversity was estimated to be 0.0055. A phylogenetic tree constructed by genetic distances among seven restriction types showed at least two clusters of mtDNA.


Endocrine cell populations in the pancreas of diabetic WBN/Kob rats. Numerical changes of insulin-, glucagon- and somatostatin-positive cells in the pancreas of WBN/Kob male rats with spontaneously occurring diabetes were examined. The rats examined were divided into three different age groups: Groups I (12 weeks old) and II (33 weeks old) were clinically prediabetic and group III (60-90 weeks old) was diabetic. Serum glucose value was in the normal range in groups I and II, while it was much higher in group III. B and A cells were markedly decreased in number in groups II and III. In group II, the ratio of B to A cells was normally preserved, though the total endocrine cell number was markedly decreased as compared with that in group I. In group III, the percentage of B cells was decreased significantly. The normal ratio in group II seemed to keep serum glucose within the normal level. In addition to the total endocrine cell reduction, an altered ratio of B and A cells was considered to cause the diabetic condition.


Acid-induced autoagglutination found in chicken pathogenic Escherichia coli strain. Chicken pathogenic Escherichia coli strains were found to autoagglutinate in a static culture of trypticase soy broth (TSB). One strain, designated PDI-386, was further studied for its autoagglutinating property. Acidity in the cultured medium caused by glucose degradation induced the autoagglutination. The bacterial cells grown in a glucose-free L-broth could be aggregated by adding acid, which suggests a potentiality of autoagglutination of the strain grown in the L-broth. The autoagglutinating parent (Agg) formed small colonies with irregular edges like rough colonies on the TS agar, whereas its non-autoagglutinating variant (Nag) formed larger smooth colonies with a perfectly round edge. The Nag colony was easily generated from the Agg colony on the TS agar. The autoagglutinating property was very unstable when the bacteria was passed in the TSB, but rather stable in the L-broth. Under electron microscope, the Agg were found to possess pili of more than 20 microns in length. However, the phenotypic expression of autoagglutination did not correlate with that of mannose-sensitive hemagglutination against guinea pig erythrocytes. Incubation of the Nag in the L-broth at room temperature for more than 10 days provoked the reversion of the autoagglutination. There was no difference between the Agg and the Nag in terms of surface hydrophobicity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of membrane proteins and LPS, and plasmid profiles. The virulence of the Agg was higher than that of the Nag. The autoagglutination property is, however, so unstable that the pathogenicity of E. coli isolates from chickens should be carefully evaluated.


Functional analysis of the promoter region of a nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L. The 5'-flanking region of gln-gamma, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements using a series of 5' deletions and hybrid gln-gamma:: CaMV 35S promoters. The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems. In the first system, the chimaeric genes were transferred to Lotus corniculatus L. using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots. In the second system, the constructs were electroporated into tobacco mesophyll protoplasts. The results of the 5' deletion analysis showed that the sequence between -597 and -21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements. Sequences located between -2000 and -597 were able to stimulate expression in nodules but not protoplasts, while the region from -597 to -354 enhanced expression in both nodules and protoplasts. Results obtained with the hybrid gln-gamma::35S promoters showed that two overlapping restriction fragments (-516/-343 and -474/-293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner. Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between -516 and -446, and their possible role in regulating gln-gamma expression is discussed.


The action of interferon alpha on human carcinoid tumours. The action of IFN-alpha has been studied in patients with carcinoid tumours. More than 300 patients have been treated with IFN-alpha for long periods of time (median 2.5 years). IFN-alpha exerts many pleiotrophic effects in carcinoid tumours. Antiproliferative effects are manifest mainly by a cell cycle block in GO-G1 phase and prolongation of the S-phase. Hormone synthesis is impaired with reduced circulating hormone levels after IFN-alpha therapy and the mechanism includes reduction of mRNA expression for various hormones. Induction in vitro of the nuclear enzyme 2'-5-A synthetase in tumour cells correlates with biochemical response and might account for reduced mRNA expression. IFN-alpha induces significantly increased intratumoral fibrosis in carcinoid metastases without significant changes in tumour size. Finally IFN-alpha causes alteration of the major histocompatibility complex (MHC) with increased expression of class I antigens on the tumour cells. The net result of all these effects of IFN-alpha is an antitumour effect in 70-80% of carcinoid tumour patients with biochemical control and abrogated tumour growth for extended periods of time. However, when IFN-alpha therapy is withdrawn tumour progression occurs within 3-9 months, indicating a controlling but not curing effect.


Genetic linkage analysis in recombinant inbred mice of P40, a putative clone for the high-affinity laminin receptor. Recombinant inbred (RI) mice were used to genetically map sequences of a 43-kDa protein, P40, that was originally identified as a high-affinity laminin receptor. More recent data have implicated this protein in development of the retina (Rabacchi et al. Development 109: 521-531, 1990), possibly via its proposed function in protein translation (G. Brawerman, personal communication). We have identified, in Southern blots, a set of P40-related sequences in BXD recombinant inbred mouse DNA. Ten restriction fragment length polymorphisms (RFLPs) segregate among the RI strains and display strain distribution patterns (SDPs) which are linked in varying degrees to previously typed loci on Chromosomes (Chrs) 1, 3, 6, 9, 11, 14, and 19. An intronic DNA probe from an incompletely processed P40 mRNA transcript overlaps with two of these loci mapping near the cholecystokinin gene locus on the distal arm of Chr 9 and to another site on the distal arm of Chr 6, suggesting that functional genes probably reside at least at these two sites. These P40 loci comprise part of a multimember gene family that is well dispersed in the mouse genome.


Synaptic excitation mediated by glutamate-gated ion channels. Excitatory synaptic transmission in the central nervous system relies predominantly on stimulation of L-glutamate-gated ion channels in postsynaptic membranes. Activation of these channels not only mediates millisecond to millisecond signalling but can also have long term influences on synaptogenesis and synaptic plasticity. Recent work has resolved some longstanding problems involving the identity of the transmitter, the postsynaptic localization of the receptor subtypes, and the time course of the transmitter in the synaptic cleft.


Nitric oxide and neurons.

Nitric oxide and neurons. In the past 2 years powerful evidence has emerged to suggest that nitric oxide functions as a neurotransmitter in both the central and peripheral nervous systems. Recent evidence suggests that it may play a role in mediating forms of synaptic plasticity such as long-term potentiation in the CA1 region of the hippocampus, and long-term depression in the cerebellum. Abnormal secretion of nitric oxide may be responsible for the neurotoxicity mediated by NMDA receptors that results in the pathophysiology of strokes and neurodegenerative diseases.


Signal transduction and regulation of neurotrophins. Our understanding of the molecular nature of neurotrophic interactions has been greatly enhanced by the recent isolation and characterization of several new neurotrophic factors and their receptors. Neurotrophic factors have been found to be regulated by neuronal activity in the central nervous system, and may be involved in activity-dependent processes throughout development and maturity.


Relationship of inherent resistance to doxorubicin, proliferative activity and expression of P-glycoprotein 170, and glutathione S-transferase-pi in human lung tumors. This study analyzed whether proliferative activity and expression of P-glycoprotein 170 (P-170) or glutathione S-transferase-pi (GST-pi) in human non-small cell lung carcinomas (NSCLC) represent independent factors in resistance to doxorubicin or whether an association exists between these factors. Thirty-six patients with previously untreated NSCLC participated in the study. The thymidine labelling index (TLI) was measured for detection of proliferative activity, and immunohistochemistry was used for detection of P-170 and GST-pi. The resistance of tumors was determined in vitro by the nucleotide incorporation assay. Negative correlations between TLI and P-170 (P = 0.0009) or GST-pi (P = 0.007) were found. Positive correlations existed between resistance to doxorubicin in vitro and P-170 (P = 0.005) or GST-pi (P less than 0.0001). Expression of P-170 and GST-pi showed highly significant (P less than 0.0001) positive correlation. The results demonstrate that a significant positive relationship between P-170 and GST-pi in NSCLC exists and that the expression is increased in resistant tumors and in tumors with a low proliferative activity.


Significance of c-erbB-2 amplification and DNA aneuploidy. Analysis in 78 patients with node-negative breast cancer. Amplification of the c-erbB-2 protooncogene and DNA aneuploidy have been reported to correlate with poor patient prognosis in human breast cancer. Several studies have investigated the prognostic value of these two factors in heterogeneous populations of patients with node-positive and node-negative disease. This study evaluated, on a series of patients with node-negative disease, whether c-erbB-2 proto-oncogene amplification and cellular DNA content could identify a subset of patients who, without adjuvant therapy, are destined to experience a relapse. Paraffin-embedded tissues of 78 patients were evaluated for cellular DNA content using flow cytometric analysis. Amplification of c-erbB-2 was determined on the same group of patients using slot-blot hybridization. The majority of patients were matched with control subjects for the following five clinicopathologic criteria: size of primary tumor, menopausal status, estrogen receptor, anniversary year of initial treatment, and age at treatment. Long-term follow-up (5-16 years) was available for each patient, none of whom received any form of adjuvant therapy. The presence of an abnormal DNA stemline was found in 47% (37 of 78) of the tissue specimens, whereas only 10% (8 of 78) of the tumors expressed from 3-fold to 22-fold c-erbB-2 amplification. Combined c-erbB-2 amplification and DNA aneuploidy occurred in a small group of patients (n = 4), all of whom experienced relapse. The four remaining tumors having excessive gene copy numbers had a diploid DNA distribution. The results indicate that tumors that overexpress the c-erbB-2 proto-oncogene have variable amounts of DNA and that c-erbB-2 amplification and DNA ploidy analysis provide limited predictive information of relapse in patients with node-negative breast cancer. Although the combination of c-erbB-2 amplification and DNA aneuploidy may be a predictor of poor prognosis in a small number of patients, neither measurement alone is effective in identifying patients at increased risk of recurrence of disease.


Hormone-regulated apoptosis results from reentry of differentiated prostate cells onto a defective cell cycle. Castration initiates extensive apoptosis of the secretory epithelial cells lining the ducts of the rat ventral prostate, resulting in the striking regression of this male sexual accessory tissue. We had previously described the paradox of finding similar cascades of gene activity (c-fos greater than c-myc greater than hsp-70) induced during the early period of ventral prostate regression and during the regrowth of the ventral prostate gland initiated by testosterone replenishment. This common pattern of protooncogene expression during periods of predominant cellular apoptosis or proliferation caused us to examine further the possibility that the two cellular events occur through identical early molecular pathways. In the present study we demonstrate that apoptotic prostate epithelial cells incorporate bromodeoxyuridine into nuclear high-molecular-weight DNA prior to nuclear DNA fragmentation. The DNA synthetic activity occurs in coordination with a massive induction of proliferative cell nuclear antigen, a proliferation marker, in the nuclei of androgen-deprived prostatic epithelial cells. Moreover, this activity is also associated with the increased expression of mRNA encoding p53, a suppressor gene well known as a cell cycle-blocking agent. Our data indicate that quiescent (G0) prostate epithelial cells undergo apoptosis due to two sequential events initiated by testosterone depletion. The first event is the active reentry of these cells into the cell cycle. The second event is the apoptotic destruction resulting from the inability of the differentiated cells to successfully complete this cycle.


Cytogenetic alterations associated with P-glycoprotein- and non-P-glycoprotein-mediated multidrug resistance in SW-1573 human lung tumor cell lines. Multidrug resistance can be induced in mammalian cells by selection with a single cytotoxic agent. Overproduction of the energy-dependent drug efflux pump P-glycoprotein, encoded by the mdr1 gene, has been identified as the cause of one form of multidrug resistance. The molecular basis of other forms of multidrug resistance is unknown. Doxorubicin selection of the human squamous lung cancer cell line SW-1573 resulted in multidrug-resistant sublines in which a non-P-glycoprotein-mediated form of multidrug resistance precedes mdr1 expression. Here we present a cytogenetic analysis of both non-P-glycoprotein-mediated multidrug-resistant and P-glycoprotein-mediated multidrug-resistant sublines derived from SW-1573. Three independently derived non-P-glycoprotein-mediated multidrug-resistant sublines showed a heterozygous deletion of the short arm of chromosome 2 (p23-pter), whereas alterations of chromosome 7 were present in the P-glycoprotein-mediated multidrug-resistant cell lines. In one series of clonally derived P-glycoprotein-mediated multidrug-resistant sublines, mdr1 overexpression was accompanied by various markers of chromosome 7 with breakpoints at 7q22, the mdr1 gene being known to be located at 7q21.1. Our data suggest that in SW-1573 cells acquisition of non-P-glycoprotein-mediated multidrug resistance is accompanied by a specific deletion or a translocation involving the short arm of chromosome 2, whereas in the emergence of P-glycoprotein-mediated multidrug resistance a rearrangement of the long arm of chromosome 7 is a critical event.


Human T-cell leukemia virus type I or a related retrovirus in patients with mycosis fungoides/Sézary syndrome and Kaposi's sarcoma. Antibodies reactive with human T-cell leukemia virus type I (HTLV-I) proteins p19, p24, gp46, p56, and gp68 were detected in four of 27 patients with mycosis fungoides/Sézary syndrome (MF/SS) and one patient with Kaposi's sarcoma using radioimmunoprecipitation and Western blot analysis. Seroreactivity patterns to HTLV-I proteins of MF/SS sera were indeterminate or limited in comparison with sera of patients with adult T-cell leukemia/lymphoma. HTLV-I gag- and tax/rex-specific DNA was demonstrated in peripheral blood from three of the MF/SS patients and from the patient with Kaposi's sarcoma by the polymerase chain reaction. HTLV-I-specific DNA sequences were not detected in a cohort of seven seronegative MF/SS patients. The frequency of HTLV-I infection was four of 27 or 14.8% among the MF/SS patients, which is several hundredfold higher than in normal blood donors. The present data suggest a possible association of HTLV-I or a related retrovirus with mycosis fungoides/Sézary syndrome and Kaposi's sarcoma.


Monoclonal antibody MRK16 reverses the multidrug resistance of multidrug-resistant transgenic mice. Using multidrug-resistant (MDR)-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers, we determined the efficacy of human-specific anti-P-glycoprotein monoclonal antibody MRK16 in overcoming multidrug resistance in an intact animal. MRK16 alone (2 mg) did not significantly affect the WBC counts of the MDR-transgenic mice, but MRK16, as well as the F(ab')2 fragments of MRK16, led to a dose-dependent circumvention of bone marrow resistance against daunomycin, doxorubicin, vincristine, vinblastine, etoposide, and taxol. This sensitizing effect could not be enhanced by combining MRK16 with low molecular weight chemosensitizing agents such as verapamil, quinine, quinidine, or cyclosporin A. We also investigated the concept of specifically targeting and killing multidrug-resistant cells by using MRK16 coupled to Pseudomonas exotoxin (PE). MRK16-PE resulted in a dose-dependent killing of bone marrow cells in MDR-transgenic mice, whereas no bone marrow toxicity was observed in normal control mice. Administration of excess MRK16 prior to injection of MRK16-PE successfully blocked the effect of MRK16-PE. MOPC-PE, a non-MDR-related control monoclonal antibody conjugate, did not target and kill multidrug-resistant bone marrow cells in MDR-transgenic mice. Thus, these immunological approaches to reversing multidrug resistance appear to be both specific and effective.


Combined antimicrotubule activity of estramustine and taxol in human prostatic carcinoma cell lines. Estramustine (EM) and taxol, two antimicrotubule agents with distinct and apparently opposing mechanisms of action, were found to be effective in combination in the preclinical treatment of EM-resistant and sensitive, wild-type human prostatic carcinoma cell lines. Estramustine combined with 1 nM taxol (concentration 100-fold less than that measured in plasma of patients treated with taxol) produced greater than additive effects on the inhibition of cell survival of both wild-type and EM-resistant cells. When taxol was used with another microtubule-destabilizing drug, vinblastine, no significantly increased cytotoxicity was observed. Other effects on wild-type and EM-resistant cells produced by the combination of EM and taxol included (a) an increased proportion of the cells in the S phase of the cell cycle; (b) no mitotic block; and (c) an increase in the percentage of micronucleated cells from a control value of less than 1% to greater than 20% after drug treatment. Immunofluorescent microscopic analysis of the effect of this drug combination on the mitotic spindle apparatus revealed specific examples of aberrant mitotic figures, including multiple asters, cells with two distinct spindles, and tripolar spindles able to traverse mitosis and complete cytokinesis. These data provide supportive preclinical evidence for the potential development of an EM/taxol combination clinical regimen either for prostate or other cancers.


Immunoelectron microscopic localization of thiolases, beta-oxidation enzymes of an n-alkane-utilizable yeast, Candida tropicalis. The location of acetoacetyl-CoA thiolase (T-I) and 3-ketoacyl-CoA thiolase (T-III), enzymes of the fatty acid beta-oxidation system, was studied in n-alkane-grown Candida tropicalis cells by immunoelectron microscopy using a post-embedding method with colloidal gold conjugated IgG. The deposition of gold particles for T-I was detected in the microbodies and cytoplasm and that of gold particles for T-III specifically in the microbodies. The double labeling technique confirmed that T-I and T-III occurred concurrently in a microbody and T-I also in cytoplasm. These results were consistent with the biochemical data based on subcellular fractionation and indicated that the yeast beta-oxidation system operates efficiently only in the microbodies.


A comparison of esmolol and labetalol for the treatment of perioperative hypertension in geriatric ambulatory surgical patients. This is an open randomized study comparing the efficacy and safety of i.v. esmolol and labetalol in the treatment of perioperative hypertension in ambulatory surgery. Twenty-two elderly patients undergoing cataract surgery under local anaesthesia were studied. The main inclusion criteria were development of systolic blood pressure greater than 200 mmHg or diastolic greater than 100 mmHg. Esmolol was given as a bolus 500 micrograms.kg-1 i.v. followed by a maintenance infusion (150-300 micrograms.kg-1.min-1). Labetalol was given as a bolus of 5 mg i.v. followed by 5 mg increments as needed up to a maximum of 1 mg.kg-1. Esmolol and labetalol both produced reductions in systolic and diastolic blood pressure (P less than 0.05) within ten minutes of administration which lasted for at least two hours. Reduction of blood pressure by esmolol was accompanied by a decrease in HR (P less than 0.05). Two patients developed extreme bradycardia (HR less than 50 beats.min-1) and esmolol had to be discontinued. Labetalol, in contrast, induced only a moderate decrease in HR. None of the patients treated with labetalol experienced any prolonged side effects such as orthostatic hypotension. In conclusion, esmolol may produce considerable bradycardia in elderly patients when hypertension is not accompanied by tachycardia. Labetalol was easier to administer in the ambulatory setting and one-tenth the cost of esmolol.


Enzyme-linked immunosorbent assay of c-erbB-2 oncoprotein in breast cancer. Amplification or increased expression of the c-erbB-2 gene has previously been reported to be a prognostic marker for breast cancer. Gene amplification is usually measured by Southern blotting, whereas increased protein expression is usually detected by immunocytochemistry. We measured c-erbB-2 protein with an enzyme-linked immunosorbent assay (ELISA). High concentrations of oncoprotein were found in 25 of 161 (16%) primary breast cancers and in 3 of 6 (50%) breast cancer metastases. High concentrations were not found in normal breast tissue or benign breast tumors. In the primary cancers, high concentrations of c-erbB-2 protein were found more frequently (a) in estrogen receptor-negative tumors than in estrogen receptor-positive tumors, (b) in progesterone receptor-negative tumors than in progesterone-positive tumors, and (c) in axillary node-positive cancers than in node-negative cancers. Patients with tumors containing high amounts of the c-erbB-2 protein had a significantly shorter (P less than 0.001) disease-free interval and overall survival rate than did patients with low amounts. We conclude that assay of c-erbB-2 protein by ELISA is simple, rapid, and quantitative and offers important prognostic information in breast cancer.


Monoclonal antibody to MALA-2 (ICAM-1) reduces acute autoimmune nephritis in kdkd mice. Hereditary tubulointerstitial nephritis is a prominent cause of renal failure in humans. A variety of animal models utilizing immunologically induced nephritis have been developed. The kdkd congenic variant of the CBA/Ca mouse has normal kidneys at birth but develops progressive, lethal autoimmune nephritis beginning at approximately Week 8. The destruction of renal tubular epithelium in mediated by a population of antigen-specific, H-2Kk-restricted, Lyt-2+, L3T4- T cells. The present experiments demonstrate that systemic treatment with anti-ICAM-1 monoclonal antibody reduces kidney disease in kdkd mice. Anti-ICAM-1 mab localizes to inflammatory sites in the kidney and effects a significant reduction in leukocyte infiltration. Concomitantly, urine protein levels of anti-ICAM-1-treated mice are significantly reduced. The use of anti-adhesion molecule monoclonal antibodies that alter leukocyte activity and/or trafficking may be useful therapies for certain autoimmune disorders.