Autoradiographic detection of vasopressin binding sites, but not of oxytocin binding sites, in the sheep olfactory bulb. Oxytocin facilitates maternal behaviour in sheep. In the present study, we searched for the presence of oxytocin and vasopressin binding sites in the sheep olfactory bulb, a brain area which is thought to be involved in specific bond formation between the ewe and its lamb. Using in vitro autoradiography, we observed binding of tritiated vasopressin to the glomerular layer of the olfactory bulb. Competition studies performed with structural analogues and the use of a 125I-labelled linear vasopressin antagonist suggested that sites which bind vasopressin are V1 type receptors. In contrast, specific binding sites for oxytocin in the olfactory bulb could be detected neither in control females, nor in ovariectomized females treated with estradiol nor in postparturient ewes, although such sites were present in the uterus.


GABAA, GABAB, and benzodiazepine binding sites in the cerebellar cortex of the red-eared turtle (Pseudemys scripta). We used receptor autoradiography to ascertain the distribution of GABAA and GABAB binding sites in the cerebellar cortex of the red-eared turtle (Pseudemys scripta). GABAA binding sites were found in both molecular and granule cell layers with highest levels in the granule cell layer. GABAB binding sites were found at highest level in the molecular layer. Benzodiazepine binding sites were found in approximately equal abundance in both layers. Little binding of any ligand was seen in the Purkinje cell layer. Our results are similar to those found in mammals and other non-mammalian vertebrates and indicate that the organization of inhibitory pathways of the cerebellar cortex has been conserved in the course of vertebrate evolution.


Antinociception produced by receptor selective opioids: modulation of spinal antinociceptive effects by supraspinal opioids. The effect of intracerebroventricular administration of low-antinociceptive doses of selective mu- (DAMGO) or delta- (DPDPE) opioid agonists on the dose-dependent antinociceptive effects produced by intrathecal administration of sequentially increasing doses of selective mu-, delta-, or kappa-(U50,488H) opioid agonists was evaluated, in the rat, using the Randall-Selitto paw-withdrawal test. When DPDPE or U50,488H was administered intrathecally, the low doses of both intracerebroventricular DAMGO and intracerebroventricular DPDPE markedly enhanced the antinociceptive effects of both intrathecal opioids. In contrast, when DAMGO was administered intrathecally, both intracerebroventricular DAMGO and intracerebroventricular DPDPE, administered in low doses, markedly antagonized the antinociceptive effects of the intrathecal opioid. In addition, the intracerebroventricular administration of a low-antinociceptive dose of a second mu-opioid agonist, morphiceptin, antagonized the antinociceptive effects of intrathecal morphiceptin. The antagonism of the antinociceptive effects observed with spinal administration of DAMGO is dose-dependent, with the effect observed only at low doses. Furthermore, the antagonism cannot be explained by a reduction in motor deficits produced by intrathecal administration of DAMGO, because there were no differences in motor deficits, measured with an accelerating Rotarod treadmill, between intrathecal DAMGO administered as a single agent or as part of a combination regimen. The differences in antinociceptive effects obtained with the various supraspinal and spinal combinations are discussed in terms of the interactions that may occur between brainstem and spinal opioid receptor sites.


Effects of methylmercury on perineurial Na+ and Ca(2+)-dependent potentials at neuromuscular junctions of the mouse. The ability of acute application of the neurotoxicant methylmercury (MeHg) to disrupt the function of presynaptic Ca2+ and Na+ channels at intact neuromuscular junctions was examined using mouse triangularis sterni motor nerves. In Ba(2+)-containing solutions, potential changes arising from Na+ and Ca2+ channel function could be recorded from the perineurial sheath surrounding motor neurons when K+ channels were blocked by tetraethylammonium chloride and 3,4-diaminopyridine. MeHg (100 microM) reduced both Na(+)- and Ba(2+)-dependent components to block within 3-5 min at apparently equivalent rates. Time to block was approximately 7 min after exposure to 50 microM MeHg. In 2 of 5 preparations exposed to 50 microM MeHg, the Ca2+ channel-mediated component was blocked prior to the Na+ channel-mediated component. In the remaining three preparations, Na(+)- and Ba(2+)-dependent potentials were blocked at similar times. Following block by MeHg, neither perfusing the preparation in MeHg-free solutions nor increasing the intensity and/or duration of stimulus to the intercostal nerves resulted in recovery of Na+ or Ca2+ potentials. In the presence of K+ channel blockers, repetitive firing of nerves in response to a single stimulus was observed in 20-30% of the triangularis preparations; in the two preparations treated with MeHg in which repetitive firing was observed, it decreased prior to block of the stimulus-induced Na+/Ba2+ potentials. These results corroborate the results obtained in isolated synaptosomes and pheochromocytoma cells, and suggest that MeHg decreases motor nerve excitability by disrupting Na+ channel function and may block neurotransmitter release by disrupting Na+ and Ca2+ channel function.


Ethanol enhances synaptically evoked GABAA receptor-mediated responses in cerebral cortical neurons in rat brain slices. Previous intracellular electrophysiological studies on rat hippocampal brain slices have shown very little effect of acute ethanol application on synaptically evoked GABAA receptor-mediated responses recorded in CA1 pyramidal neurons. The present study was designed to compare the effects of ethanol on pyramidal neurons in the hippocampus and cerebral cortex. Using conventional intracellular microelectrodes (60-80 M omega) to impale cortical neurons in brain slices, 80 mM ethanol application did not affect the membrane input impedance nor evoked EPSPs, but significantly affected the resting membrane potential (usually a 2-5 mV hyperpolarization). When stimulus-evoked GABAA-mediated IPSCs were studied using whole-cell recordings from cortical neurons voltage-clamped at depolarizing potentials, monophasic IPSCs were evoked that were blocked by bicuculline, increased by pentobarbital, and enhanced by ethanol superfusion in a dose dependent manner over the range of 20-160 mM. Hippocampal IPSCs recorded under identical conditions were not enhanced by ethanol. Parallel studies of GABA-stimulated 36Cl- flux measurements in microsacs prepared from hippocampal, cerebral cortical and cerebellar tissue demonstrated that ethanol significantly enhanced (30-50%) 36Cl- flux in microsacs derived from the cerebral cortex and cerebellum, but not in microsacs prepared from the hippocampus. These results demonstrate that there are clear brain region-dependent differences in the way that GABAA receptor function is altered by acute ethanol, and that these differences are apparent not only as an enhancement of responses to exogenous GABA, but also as a facilitation of the responses to endogenous GABA released from inhibitory nerve terminals during synaptic activation.


Cyclic adenosine 3'5'-monophosphate potentiates excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons. Intracellular recordings were made from rat dorsal horn neurons in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). In the presence of TTX, bath application of the membrane permeable analogue of cyclic AMP, 8-Br cyclic AMP (25-100 microM) caused a small depolarization of the resting membrane potential accompanied by a variable change in membrane input resistance. In addition, 8-Br cyclic AMP caused a long-lasting increase in the spontaneous synaptic activity and the amplitude of presumed monosynaptic excitatory postsynaptic potentials evoked in the substantia gelatinosa neurons by orthodromic stimulation of a lumbar dorsal root. When the fast voltage-sensitive Na conductance was blocked by TTX, 8-Br cyclic AMP enhanced in a reversible manner, the depolarizing responses of a proportion of dorsal horn neurons to N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and kainic acid (KA). The effects of 8-Br cyclic AMP on the resting membrane potential and the NMDA response of dorsal horn neurons were mimicked by reducing phosphodiesterase activity with bath application of 3-isobutyl-1-methylxanthine, but not by cyclic AMP applied extracellularly. Moreover, we have found that intracellular application of a protein inhibitor of cyclic AMP-dependent protein kinase (PKI) into dorsal horn neurons prevents the 8-Br cyclic AMP-induced potentiation of the NMDA response of these cells. These results suggest that in the rat spinal dorsal horn the activation of the adenylate cyclase-cyclic AMP-dependent protein kinase system may be involved in the enhancement of the sensitivity of postsynaptic excitatory amino acid (NMDA, AMPA, KA) receptors and modulation of primary afferent neurotransmission, including nociception.


Ontogeny of zeta (zeta), the opioid growth factor receptor, in the rat brain. Opioid growth factor (OGF), [Met5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (zeta) opioid receptor to perform its function. Using [3H]-[Met5]enkephalin, the ontogeny of the zeta receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (E15). In the whole brain, binding capacity (Bmax) was two-fold greater at E15 than at E18 and E20. The quantity of zeta receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8-10. The Bmax of the zeta receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be detected. Continuous opioid receptor blockade from birth to postnatal day 6 increased body weight, the Bmax of the zeta receptor in the whole brain and cerebellum (but not the Kd), and increased the number of layers of germinal cells in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuropeptide Y inhibits alpha-MSH release from rat hypothalamic slices through a pertussis toxin-sensitive G protein. The arcuate nucleus of the hypothalamus contains various types of peptidergic neurons. In particular, two distinct populations of neurosecretory neurons containing neuropeptide Y (NPY)- and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity have been identified in the arcuate nucleus. Double-labeling immunocytochemical data have recently shown that NPY-containing fibers make synaptic contacts with proopiomelanocortin (POMC) immunoreactive neurons. We have thus investigated the possible effect of NPY on the release of alpha-MSH from rat hypothalamic slices in vitro, using the perifusion technique. NPY significantly inhibited KCl-stimulated alpha-MSH release in a dose-dependent manner. The inhibitory effect of NPY was mimicked by the Y2 agonist, NPY-(13-36), while the Y1 agonist, [Leu31,Pro34]NPY, was devoid of effect. Pretreatment of hypothalamic slices with pertussis toxin (PTX) blocked the inhibitory effect of NPY, suggesting that the action of NPY on POMC neurons is mediated through a PTX-sensitive G protein. These results support the notion that NPY may play a physiological role in the regulation of alpha-MSH release from hypothalamic neurons.


Neonatal lesions of the ventral tegmental area affect monoaminergic responses to stress in the medial prefrontal cortex and other dopamine projection areas in adulthood. A mean decrease of dopamine (DA) to 20% and serotonin to 25-30% of control levels was found in the medial prefrontal cortex (mPFC) and amygdala/piriform cortex (A/PC) of adult rats with neonatal lesions of the ventral tegmental area (VTA). The metabolites were less decreased suggesting an increased activity of the remaining terminals. Moderate decreases to 30-75% were detected for DA and serotonin in the nucleus accumbens, olfactory tubercle and striatum. Footshock stress in control animals resulted in a strong increase (200% of control) in DA metabolites in mPFC and A/PC. The noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) in A/PC was strongly increased to 240%. When stress was given to the neonatally VTA-lesioned animals these neurochemical responses were reduced compared to the nonlesioned rats. In the case of DA in the mPFC this was clearly due to a loss of stress response in the severe lesion group where DA is depleted to less than 20% of control. The stress-induced small increases in DA metabolism in tubercle, accumbens and striatum and serotonin metabolism in the striatum (20-40%) were entirely lost, while the MHPG increase in the A/PC was blunted. The present results suggest that moderate and severe lesions of DA and serotonin alter or prevent the normal activation of these transmitter systems and even of the noradrenergic system to stress.


Age-dependent modulation of in vivo cortical acetylcholine release by benzodiazepine receptor ligands. In vivo microdialysis was utilized to determine the effects of benzodiazepine receptor (BZR) ligands on cortical acetylcholine (ACh) release in awake young and aged rats. There were no significant differences in baseline cortical ACh release as a function of age. While administration of the BZR selective inverse agonist ZK 93 426 increased ACh release in both groups of animals, the aged rats exhibited a greater stimulation. Unexpectedly, under the present testing conditions, the BZR agonist chlordiazepoxide (CDP) had no systematic effect on ACh release in either group. The presence or absence of these drug effects or drug-age interactions was not secondary to the impact of these compounds on behavioral activity. Cortical ACh release could also be stimulated by turning off the lights in the observation room or by the systemic administration of scopolamine. Aged rats were at least as able as their younger counterparts to respond to these manipulations with increased release. These results suggest that basal and stimulated release of cortical ACh is not impaired at the ages studied. Moreover, selective inverse BZR agonists may be a potent way of trans-synaptically stimulating cortical cholinergic transmission.


Propofol potentiates the binding of [3H]flunitrazepam to the GABAA receptor complex. Propofol (2,6-diisopropylphenol) robustly stimulated the binding of 1 nM [3H]flunitrazepam (FNZ) to rat brain membranes with an EC50 of 146 microM in chloride-free buffer and 23 microM in buffer containing 200 mM NaCl. NaCl showed an EC50 of 40 mM for its ability to increase the potency of propofol. The ability of a range of anions to potentiate propofol's interactions with the GABAA-benzodiazepine receptor was closely correlated with their permeabilities at this ion channel. Propofol, at a concentration of 300 microM, decreased the EC50 for the potentiation of FNZ binding by NaCl from 39 mM to 13 mM, with no change in the maximal potentiation. At a concentration of 30 microM, propofol significantly decreased the EC50 for potentiation of FNZ binding by the neurosteroid alphaxalone whilst increasing that for potentiation by pentobarbitone. We conclude that propofol is a potent barbiturate-like modulator of [3H]flunitrazepam binding.


The time course of changes in D1 and D2 receptor binding in the striatum following a selective lesion of striatonigral neurons. The suicide transport agent volkensin was used to produce a selective lesion of striatonigral projection neurons and the time course of changes in binding at striatal D1 and D2 receptors analyzed. Both show a time-dependent decrease with two-thirds of the total change occurring within the first 10 days and a greater decrease in D1 receptor density at all time points. Our results confirm selective localization of D1 receptors to striatonigral neurons and are consistent with localization of some striatal D2 receptors to striatonigral neurons.


[Recombinant colorimetric antibodies: genetic construction and production in E. coli]. We constructed an expression vector comprising a transcription unit composed of (1) the gene of alkaline phosphatase from E. coli in which we inserted a DNA fragment encoding the VH and CH domains of an IgG2A; (2) a DNA fragment encoding the light chain of the same immunoglobulin. Bacteria, E. coli were transformed with the plasmid, and hence it produced a periplasmic and chimaeric protein having both the alkaline phosphatase enzymatic activity and the immunoglobulin binding function. The hybrid protein mimics a bivalent antibody whose Fc part is replaced by a dimer of alkaline phosphatase. Recombinant colorimetric antibodies offer interesting potentialities for future developments in the field of immuno-enzymatic diagnosis.


Effects of cytokines on human basophil chemotaxis. Basophil chemotactic activity (BCA) of eight recombinant human (rh) cytokines was examined. Highly purified basophils were obtained by Percoll discontinuous gradients, followed by negative selection using flow cytometry. Then BCA was measured by means of modified Boyden chamber method. Both interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) had much more potent BCA than complement C5a, leukotriene B4 and platelet activating factor, well known as granulocyte chemotactic factors. Chemotaxis rather than chemokinesis was shown in chequerboard analysis of basophil migration induced by IL-3 and GM-CSF. Relatively high concentrations of IL-5 also induced basophil migration, although predominantly chemokinetic. IL-8 had apparent BCA, which was not so high as that of C5a. In contrast, IL-2, IL-4, interferon(IFN)-gamma and granulocyte colony-stimulating factor (G-CSF) had no significant BCA. These findings suggest that IL-3, IL-5, GM-CSF and, perhaps, IL-8 have an effect on basophil migration as well as modulation of basophil mediator release and may provide some insight into the basophil accumulation observed in late-phase allergic responses.


Anti-inflammatory effects of nedocromil sodium: inhibition of alveolar macrophage function. The IgE-dependent activation of mononuclear phagocytic cells through their capacity to express low affinity IgE receptors (Fc epsilon RII) has been proposed as a mechanism for the development of airways inflammation in allergic asthma. This Fc epsilon RII expression leads to the IgE-dependent production of the potent pro-inflammatory cytokines IL-1 beta and TNF-alpha. Expression by monocytes of Fc epsilon RII is regulated by several cytokines including interleukin-4, gamma- and alpha-interferons, and granulocyte-macrophage and macrophage colony stimulating factors. An anti-inflammatory effect of nedocromil on monocytes has been proposed as a possible mechanism for its anti-asthma activity. We therefore investigated the capacity of nedocromil to modulate mononuclear phagocyte Fc epsilon RII expression and cytokine production. We used an anti-Fc epsilon RII antibody and flow cytometric analysis to assess the capacity of nedocromil to modulate cytokine-induced Fc epsilon RII expression in normals and asthmatics. Monocytes, THP-1 monocyte leukaemia cells, and alveolar macrophages were exposed to varying concentrations of these cytokines for 48 hr at 37 degrees C with or without the additional presence of nedocromil (1-10 microM) and the per cent of monocytes expressing Fc epsilon RII was determined. No changes in Fc epsilon RII expression were observed. Subsequently, we investigated the capacity of nedocromil to affect the capacity of IgE plus anti-IgE complexes, allergen, and LPS (16 hr/37 degrees C) to stimulate IL-1 beta and IL-6 production. No changes were observed when nedocromil was applied concomitant with the stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-term efficacy of percutaneous transluminal coronary angioplasty. Percutaneous transluminal coronary angioplasty (PTCA) is a well-accepted procedure used in therapy for coronary artery stenosis. It is a delicate procedure depending upon the operator's experience and technique. Major efforts have been directed toward improving such performance. This is a report of our growing experience accumulated over the last eight years. From July 1983 to December 1990, 893 cases of coronary artery disease with a total of 1111 procedures received PTCA in this hospital. The success rate in dilatation increased year by year; to peak in 1990 (94%). PTCA was performed in 447 cases of single vessel disease; 310 cases of double vessels disease; and 136 cases of triple vessels disease. Dilated coronary artery lesions included left anterior descending artery, 637 (64%), right coronary artery, 198 (20%), and left circumflex artery 157 (16%). More than one procedure was undertaken in 178 patients, and about 20% patients had repeated PTCA. Major complications such as acute myocardial infarction developed in 3.0%. One point one percent had emergency surgery and the mortality rate was 1.8%. The majority of cases were followed up and appeared well after successful angioplasty. In conclusion, PTCA is an valuable procedure for treatment of critical coronary artery stenosis, with acceptable complications.


Community-based survey on blood pressure, blood biochemistry and dietary habits in Pu-Li, Taiwan. The target population of this study were all residents over the age of 30 in Pu-Li. Stratified cluster sampling was employed. Household visits were carried out in 1987 by the Yang-Ming Crusade, organized by well-trained medical students of the National Yang-Ming Medical College. A total of 1,738 persons out of a potential 2,573 eligible subjects were contacted by at least one of the two interviews or a blood draw. The crude and adjusted (for vacant households) rates were 68% and 84% respectively. Age, sex, and education were evenly distributed. The distribution of physiological variables and blood biochemistry were as follows: 23.0 +/- 3.2 for body mass index (BMI = kg/m2; men 22.8 +/- 3.0, women 23.2 +/- 3.3), 129.7 +/- 20.7 mmHg for systolic blood pressure (men 130.4 +/- 19.6 mmHg, women 128.9 +/- 21.8 mmHg), 81.9 +/- 12.9 mmHg for diastolic blood pressure (men 82.9 +/- 12.8 mmHg, women 80.9 +/- 12.9 mmHg), 103.6 +/- 29.6 mg/dl for fasting plasma glucose (men 101.8 +/- 21.9 mg/dl, women 105.2 +/- 35.2 mg/dl), 222.1 +/- 57.2 mg/dl for cholesterol (men 221.9 +/- 56.5 mg/dl, women 222.3 +/- 57.9 mg/dl), 162.7 +/- 115.5 mg/dl for triglyceride (men 170.3 +/- 117.1 mg/dl, women 155.7 +/- 113.6 mg/dl), 5.8 +/- 3.0 mg/dl for uric acid (men 6.5 +/- 4.8 mg/dl, women 5.2 +/- 1.5 mg/dl). Blood pressure and blood biochemistry variables were positively associated with age. Comparatively, men had much more unhealthy life styles than women; due to prevalences for cigarette smoking (56.0% vs. 3.0%), alcohol intake (43.8% vs. 6.6%) and betel-nut chewing (18.2% vs. 0.8%).(ABSTRACT TRUNCATED AT 250 WORDS)


Does Hakka ethnic group have higher incidence of thalassemia traits in Taiwanese population? The purpose of this survey is to find out whether Hakka group has higher incidence of thalassemia traits in our population. A total of 1,115 healthy employees from a company were screened by complete blood count (CBC) with indices. Those subjects with mean corpuscular volume (MCV) less than 80 fl were further evaluated by hemoglobin electrophoresis and modified hemoglobin H (Hb H) inclusion staining to confirm the diagnosis of beta- and alpha-thalassemia traits, respectively. We evaluated and compared the crude occurrence rates of thalassemia traits in Hakka, non-Hakka, and Taiwanese. Subjects with one or both Hakka parents had higher crude incidence of alpha-thalassemia traits than other groups of subjects, but this phenomenon wasn't found in beta-thalassemia traits.


An evaluation of long-term nasal CPAP therapy for sleep apnea. Nasal continuous positive airway pressure (CPAP) is an effective therapy for sleep apnea. Little is known about long-term patient compliance and side effects with this therapeutic modality in the Chinese. In order to evaluate this, we collected 8 obstructive sleep apnea patients who received home nasal CPAP therapy between January 1990 and July 1991. Each received two sets of nap polysomnographic studies. The initial set was performed to diagnose and evaluate patient response to CPAP as well as defining the CPAP pressure the patient would be using at home. The second set of studies were conducted for follow up and re-evaluation. Seven of these patients reported using nasal CPAP during sleep at night, and one did not use it all. Nasal CPAP improved clinical symptoms, particularly daytime sleepiness, and 7 patients were generally satisfied with nasal CPAP. Initially the side effects were a dry throat and nose. After 5 to 15 months of CPAP treatment, the follow-up nap sleep studies showed no significant change in the apnea/hypopnea index, duration of apnea, or oxygen desaturation between the diagnostic and follow-up (without CPAP) studies. However the amount of nasal CPAP pressure setting declined in 4 of 7 patients. Our own experience indicates that long-term nasal CPAP is an important new means of treatment for sleep apnea and allows a normal daytime life. It was well-tolerated by most sleep apnea patients. However, it is necessary to further evaluate of morbidity and the amount of pressure setting relative to long-term home nasal CPAP.


Surgical treatment of diaphragmatic eventration in adults. Surgical intervention of diaphragmatic eventration is urgent and life-saving in infants but is rarely in adults. In the past 22 years, Seven adults with diaphragmatic eventration were encountered at the Division of thoracic surgery, Dept. of surgery, VGH, Taipei. The age on diagnosis ranged from 32 to 65 years with mean of 44 years old. Male and left diaphragm were more frequently affected (6 men, 1 woman; 6 left, 1 right). All of them received operation of plication. In spite of good immediate postoperative return of the diaphragms to normal position with clinical improvement found in all patients, 5 dyspneic patients were found to have gradual diaphragmatic rise or relapse of respiratory symptoms after repair. Diaphragmatic eventration could cause compression of lung by abdominal organs and reinforcement of diaphragm through plication might increase the diaphragm strength and diminish the clinical symptoms. Yet according to our series, we recommend surgical intervention is only for existence of distress stemmed from it and unresponsive to medical therapy.


Correlation between sonographic and angiographic findings of extracranial carotid artery disease. Comparison between sonographic and angiographic findings of the extracranial carotid arteries were made in 68 patients. We used a Diasonic Duplex Ultrasonograph (DRF 400) which combined a real-time B-mode imaging system and a pulsed-wave Doppler spectral analyser. Continuous-wave Doppler was also used to determine ophthalmic artery flow direction. The findings were classified into four categories: (1) normal or less than 10% stenosis, (2) 10-49% stenosis, (3) 50-99% stenosis, (4) and total occlusion. The agreement between the two tests was 0.53 which meant fair agreement beyond chance by Kappa statistics. When angiograms were used as gold standard, the sensitivity of duplex sonography was above 80% in all categories of stenosis, and the overall accuracy and specificity were above 90% in those stenosis greater than 50% and occlusion. We conclude that duplex sonography, a reproducible noninvasive test, is a safe and cost-efficient screening method which can provide a good correlation with angiograms in detecting extracranial carotid artery disease.


[An opinion survey of physicians on the impact of National Health Insurance]. In order to further protect the rights of citizens to health care, the government of Taiwan has decided to fully implement the National Health Insurance (NHI) program by 1994. This study examined the opinions of physicians in Taiwan on the impact of the NHI. The data for the analysis came from a mailing survey of 1619 physicians carried out in November 1989 (response rate 21.6%). The results of this study showed: (1) The impact of NHI on the profession: physicians expect their incomes to decrease, workloads to increase, and their professional authonomy to decrease; (2) The impact of the NHI on working conditions: hospital-based physicians expect to decrease the use of medical auxilliary personnel, increase the volume of services, and increase service hours, where as office-based physicians expect the NHI to increase the use of medical auxilliary personnel, increase the volume of services, and increase administrative workload; (3) Half of the physicians expect the NHI will cause the quality of health services to deteriorate. Based on the above findings, this study further discussed the policy and research implications for planning the NHI.


[Bone mineral density in patient with renal transplant]. Progressive renal failure may cause disturbance of mineral metabolism. Successful renal transplantation may correct many features of disturbance of calcium and phosphorus metabolism. Dual photon absortiometry (DPA) provides a noninvasive method for the serial measurements of bone mass of the vertebral bodies. Using this method, bone mineral density (BMD) was detected and evaluated in patients with renal transplants. In this study, BMDs of lumbar vertebrae 2 to 4 were measured and the mean value was presented as gm/cm2. Forty seven patients (over 40 years of age) with renal transplants and 103 normal controls of a similar age group were included in this study. Surprisingly, no significant difference in bone mineral density was found between the patients with renal transplant and the normal controls in our preliminary study.


[Comparison of nicardipine and nifedipine in treatment of Chinese senile hypertension placebo-control, double-blind, randomized and crossover study]. The purpose of this placebo-control, double-blind, randomized and crossover study is to evaluate the effect of nicardipine and nifedipine in Chinese senile hypertension. Among totally 37 senile hypertensive patients enrolled, 26 patients (25 males, 1 female) from 55 to 78 years of age (mean 65) who had finished one part or whole protocol were studied. Totally 18 cases after 6-week treatment of nicardipine (Perdipine) had blood pressure decrease significantly from 152.6 +/- 12.3/99.6 +/- 5.7 to 140.4 +/- 15.6/93.8 +/- 8.1 mmHg in supine position (P < 0.05), and from 153.3 +/- 12.7/98.7 +/- 7.7 to 139.2 +/- 13.5/90.7 +/- 7.6 mmHg in standing position (p < 0.05). Twenty-five cases after 6-week treatment of nifedipine (Towarat) also had significant blood pressure decrease from 155.0 +/- 13.3/99.5 +/- 8.4 to 144.2 +/- 10.0/95.3 +/- 9.2 mmHg in supine position (P < 0.05), and from 151.5 +/- 17.8/100.6 +/- 9.5 to 138.6 +/- 12.8/90.4 +/- 8.3 mmHg in standing position (p < 0.05). Heart rate was unchanged in both groups. Both nicardipine and nifedipine decreased blood pressure and increased heart rate significantly with the first dose of medication in the morning (P < 0.05). There was 6.5% and 9.0% decrease of systolic and diastolic blood pressure with nicardipine in supine position, 10.1% and 11.2% decrease with nifedipine in supine position, 6.3% and 7.2% decrease with nicardipine in standing position, and 9.7% and 10.6% decrease with nifedipine in standing position. The major side-effects were palpitation (20%) and lower abdominal distension (16%) with nicardipine; and nausea or vomiting (22%) and dizziness (15%) with nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)


Enterogenous cyst of the fourth ventricle: case report. The authors present an intracranial enterogenous cyst of the fourth ventricle in a Chinese woman having symptoms of headache, dizziness and vertigo for approximately six months. A computerized tomography study of the brain disclosed a low density lesion within the fourth ventricle. A cystic tumor was completely removed and a histopathological examination diagnosed it is an enterogenous cyst; a rare lesion. During the post-operative follow-up, her symptoms gradually resolved and she was free of complaints within two years.


Thyrotoxicosis accompanied with periodic seizure attacks a case report and review of literature. A 58-year-old female was admitted and discovered to be a victim of thyrotoxicosis. She had experience periodic seizure attacks for 14 months. These seizures disappeared when function of the thyroid returned to normal. We performed many studies to search for the cause of the seizures. No epileptic focus could be detected from an EEG or a MRI of the brain. There was no abnormal laboratory data such as: hypoglycemia, hypoxemia, serum electrolyte imbalance, or an acid-base imbalance. No evidence of CNS infection was noted. She had good response to antithyroid treatment. We therefore suggest, that the seizure attacks may be related to thyrotoxicosis. In reviewing the literature, we found that only 13 cases of thyrotoxicosis with seizures have been reported since 1956 [1-9].


Successful management of perforating injury of right atrium by chest tube. Tube thoracostomy is an invasive procedure caring about 1% complication rate. Most confronted complications include diaphragm or lung laceration [1-3], damage to intrabdominal organ, intercostal artery bleeding and unilateral pulmonary edema. Here we present another rare complication of perforation of the right atrium which occurred in a patient with rheumatic heart disease (RHD) who had received mitral valve replacement (MVR) and tricuspid annuloplasty. Severe tricuspid regurgitation (TR) with huge right atrium was noted. Chest tube was inserted for pleural effusion drainage. The lesion was proved to be in the right atrium by echocardiography and computerized tomography three days later. The penetrated hole was repaired with bovine pericardium patch with minimal blood loss. This case attests to the extreme caution warranted when performing tube thoracostomy in patient with huge cardiomegaly.


Primary malignant melanoma of the vagina and cervix uteri: case report and literature review. Malignant melanoma of vagina is rare. Malignant melanoma of the cervix is even rare. To date, only 96 cases of the former and 26 cases of the latter have been reported. We report here a very rare case of primary malignant melanoma involving both vagina and cervix in a 70-year-old woman. The clinical, cytologic, light microscopic and ultrastructural findings are presented, and the pertinent literature is reviewed and discussed.


[Multiple leiomyosarcomas of extremities]. Leiomyosarcoma is an uncommon tumor that rarely occurred in extremity. Multiple leiomyosarcomas in different extremities is even rarer. We report a 90 year-old male veteran who was found to have four leiomyosarcomas, one in right arm and the other three in right thigh. The patient sustained gunshot injuries over right thigh and right arm about 60 years ago in a war with severe wound sepsis due to open communited fracture of the femur. These tumors happened to distribute in the vicinity of the old scars. It was speculated that these multicentric tumors were due to decreased immune surveillance caused by aging and induced by the previous trauma. The literatures were reviewed and the diagnostic methods, surgical treatment and the adjuvant therapy were discussed.


Synthesis of analogues of myo-inositol 1,4,5-trisphosphate that contain sulfonamide, sulfate, methylphosphonate, and carboxymethyl groups. The syntheses are described of four isosteric racemic myo-inositol 1,4,5-trisphosphate (1) analogues with the phosphate groups replaced by sulfonamide (2), sulfate (3), methylphosphonate (4), and carboxymethyl (5). None of these compounds had any affinity for the IP3 receptor or induced platelet aggregation.


Nucleoside cyclic 3',5'-phosphates: chair-twist equilibria of the phosphate rings of methyl phosphate and phenylphosphonate derivatives of cTMP. The cis and trans forms of thymidine methyl cyclic 3',5'-phosphate (9) and of the corresponding phenylphosphonate derivatives (10) were prepared in two steps from the corresponding cyclic amide. 1H NMR spectroscopy showed the phosphorus-containing six-membered rings of cis-9 (MeO and thymin-1-yl cis) to be in the chair conformation, but trans-9 to be approximately 40% in the twist conformation in pyridine-d6 and 33% in acetone-d6. For trans-9, there was resistance of conversion of the chair form into the twist form with the MeO group pseudo-axial with a delta G degrees(C-->T) value of 3.0 kcal/mol. Similarly, for cis-10 (23-39% twist population in CD3CN, acetone-d6, and CDCl3), a delta G degrees(C-->T) value of 0.5-0.6 kcal/mol for placement of the phosphoryl oxygen pseudo-axial was obtained. Both values are taken to be approximations for delta G degrees(C-->T) for cTMP. For trans-10, the twist conformation was populated to the extent of 6-13% with a delta G degrees(C-->T) value of 1.3 kcal/mol.


Derivatives of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate that mimic the actions of adenosine 3',5'-phosphate (cAMP) and guanosine 3',5'-phosphate (cGMP). A series of new analogues of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate (cBIMP) has been designed according to the properties predicted by the MNDO method, and synthesised from substituted benzimidazoles. Dipole vectors and HOMO and LUMO energies for each benzimidazole base were calculated by the MNDO method and the lipophilicities of the cBIMP derivatives were determined. In general, the cBIMP derivatives activate cAMP-dependent protein kinases I and II and preferentially bind to site B, especially for the type II kinase, with 2-trifluoromethyl-cBIMP and 5,6-difluoro-cBIMP exhibiting the highest site selectivity. Each cBIMP derivative can stimulate cGMP-stimulated cyclic phosphodiesterase (cGS-PDE), with 5,6-dimethyl-cBIMP being as potent as cGMP, and also inhibit cGMP-inhibited phosphodiesterase (cGI-PDE). Only the 2-trifluoromethyl-cBIMP and the Rp-phosphorothioates (cBIMPS) (equatorial P = S) were resistant to hydrolysis by cPDE. The Sp-phosphorothioates were hydrolysed slowly, if at all. In addition to exhibiting a high lipophilicity, the most active compounds for the induction of apoptosis and inhibition of proliferation were also resistant to cPDE (Sp-5,6-dichloro-cBIMPS) and/or were potent activators of cAMP-dependent protein kinase (5,6-dichloro-cBIMP).


The role of the 2- and 3-hydroxyl groups of 1D-myo-inositol 1,4,5-trisphosphate in the mobilisation of calcium from permeabilised human 1321N1 astrocytoma cells. The functional significance of the 2- and 3-hydroxyl groups of 1 D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was probed by using Ins(1,4,5)P3 analogues variously modified at positions 2 and 3 or elsewhere. The intrinsic activities of these compounds were compared to that of Ins(1,4,5)P3, using the calcium-mobilising receptor of the 1321N1 human astrocytoma cell line. The ligand-binding affinities were also determined using membrane preparations from rat cerebellum and bovine adrenal cortex. The results show that HO-2 and HO-3 of Ins(1,4,5)P3 have a relatively insignificant role in receptor binding and calcium release. However, the possibility of a regulatory role for the 3-position of Ins(1,4,5)P3 in these processes is proposed.


Viability of Streptococcus mutans and Streptococcus sobrinus in whole saliva with varying concentrations of indigenous antimicrobial agents. We have studied the possible relationship between indigenous salivary antimicrobial agents, indigenous mutans streptococci and the capability of added mutans streptococci to grow in saliva. Stimulated whole saliva was collected from 19 healthy donors. Saliva samples were sterilized, supplemented with glucose and inoculated with Streptococcus mutans or Streptococcus sobrinus. The mixtures were incubated for 20 h followed by counting of viable cells. Saliva samples were analysed, both before and after sterilization, for indigenous antimicrobial agents and the bacterial flora. The subjects could be divided into two groups: those (n = 9) whose saliva promoted and those (n = 10) whose saliva inhibited the growth of the inoculated streptococci. A statistically significant correlation (+0.82, p < 0.001) was found between the numbers of viable cells of S. mutans and S. sobrinus after incubation in saliva. The sterilization procedure reduced the content of all antimicrobial proteins. Salivary antimicrobial factors, or levels of indigenous mutans streptococci, did not differ between the two groups. We conclude that none of the individual salivary antimicrobial factors alone can explain the large individual differences in growth-promoting or growth-inhibiting patterns of saliva on S. mutans and S. sobrinus. Inter-individually, saliva either supports or inhibits the growth of mutans streptococci, indicating a similar response of these two species in relation to the properties of saliva.


Degradation of Met-enkephalin by hemolymph peptidases in Mytilus edulis. 1. Met-enkephalin is degraded by peptidases present in the hemolymph fluid and hemocyte membrane suspension of Mytilus edulis. Degradation of Met-enkephalin is rapid in the fluid and slower in the membrane. 2. Aminopeptidase activity is bestatin sensitive in hemocyte membrane and highest in the fluid of the hemolymph, which appears to have a component which is insensitive to inhibitor. 3. ACE activity is found only in the fluid of the hemolymph. 4. Carboxypeptidase and NEP (CD10: "enkephalinase") are membrane bound and the former appears to predominate. Phosphoramidon inhibits not only NEP, as expected, but the invertebrate carboxypeptidase as well.


Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation by dexamethasone, isoproterenol, or prostaglandin E2 either alone or in combination. 1. The purpose of these studies was to investigate the modulation of the proliferation of human T cells obtained from peripheral blood by dexamethasone (DEX), isoproterenol (ISO), and prostaglandin E2 (PGE2). The former two substances interact with T cells via the glucocorticoid and beta-adrenergic receptors respectively. When occupied by their natural ligands, glucocorticosteroids and catecholamines, these receptors have a role in modulating T-cell function during stress. During the inflammatory response increased levels of PGE2 bind to their receptors on T cells and thus alter responsiveness. Proliferation of T cells was induced by immobilized anti-CD3 monoclonal antibody (mAb) in the presence or absence of an additional costimulatory signal delivered by anti-CD28 mAb. 2. Various physiologic concentrations of DEX, ISO, or PGE2 were added at the time of initiation of the cultures and subsequent proliferation of the unstimulated T cells was determined. The results demonstrate that physiologic concentrations of all three of these agents inhibit the anti-CD3 mAb-induced proliferation of T cells. 3. Although DEX and PGE2 were equipotent in suppressing T-cell proliferation, ISO was much less effective. 4. Because concomitant elevations in the peripheral levels of these substances may occur, experiments were performed to determine the T-cell inhibitory effects of DEX together with either PGE2 or ISO. Synergistic suppression of T-cell proliferation was observed when various concentrations of DEX and PGE2, but not DEX and ISO, were added to cultures. This synergistic suppression could not be explained by an increase in cAMP accumulation in T cells stimulated with DEX and PGE2. 5. Finally, the addition of anti-CD28 mAb to anti-CD3 mAb-stimulated T cells overcame much of the suppression of proliferation induced by PGE2 or ISO but less so than that induced by DEX.


Corticotropin-releasing factor-induced immunosuppression in human and invertebrate immunocytes. 1. Corticotropin-releasing factor (CRF) appears to be a potentially important signal molecule in both vertebrate and invertebrate neuroimmune and autoimmunoregulatory processes. It appears to mimic the effects of alpha-melanocyte stimulating hormone (MSH) but has a longer duration of action. 2. alpha-Helical CRF, a specific inhibitor of CRF, antagonizes CRF-induced cellular immunosuppression but is ineffective in altering MSH-induced immunosuppression. 3. Both human and Mytilus immunocytes appear to have specific CRF receptors. 4. In another experiment, both CRF and MSH antagonize tumor necrosis factor stimulation of immunocytes. Again, alpha-helical CRF antagonizes only CRF activity, further suggesting the presence of a separate CRF receptor on these cells.


Comparison of the efficacy and tolerability of Prinivil and Procardia XL in black and white hypertensive patients. The efficacy and tolerability of Prinivil and Procardia XL were compared in 135 (67 black, 68 white) patients with mild to moderate uncomplicated essential hypertension. The goal of therapy was to achieve and maintain a supine diastolic blood pressure (SDBP) of > 90 mmHg or a decrease in SDBP > or = 10 mmHg. Patients received Prinivil 10 to 40 mg once daily or Procardia XL 30 to 120 mg once daily during a titration period of 2 to 8 weeks to achieve the goal blood pressure before a 4-week maintenance period. The mean baseline supine systolic/diastolic blood pressures were 151/97 mmHg in patients receiving Prinivil and 153/99 mmHg in patients receiving Procardia XL. Ninety-one percent of patients receiving Prinivil and 95% of those receiving Procardia XL achieved SDBP control at the end of titration therapy. At the end of the maintenance treatment period, 79% of patients receiving Prinivil and 80% of those receiving Procardia XL had SDBP control. Mean decreases in SDBP from baseline were comparable for both treatment groups. At the end of the titration period, mean decreases were 9.6 mmHg in patients receiving Prinivil and 11.3 mmHg in patients receiving Procardia XL; at the end of the maintenance period, mean decreases were 10.8 mmHg and 12.1 mmHg, respectively. There were no differences in treatment responses in either the black or white hypertensive subgroups. Thus both drugs were equally effective in black and white patients with mild to moderate essential hypertension. Both drugs were generally well tolerated, but the number of adverse experiences requiring discontinuation of therapy was significantly higher (P = 0.03) in patients receiving Procardia XL.


Modulation of inositol(1,4,5)trisphosphate-sensitive calcium store content during continuous receptor activation and its effects on calcium entry. Changes in intracellular Ca2+ concentration ([Ca2+]i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40-48 h. In these cells, the carbachol-induced sustained increase in [Ca2+]i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca2+ and a corresponding further enhancement of Ca2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P3 or changes in the generation of Ins(1,3,4,5)P4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P3, or a desensitization of the Ins(1,4,5)P3 receptor/Ca(2+)-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca2+ entry specifically on the status of the Ca(2+)-uptake component of the agonist-sensitive store, rather than the Ca(2+)-release component.


Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry. We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.


Testosterone sensitive dihydropyridine binding in the Harderian gland of the male hamster. It is well known that in different tissues, dihydropyridines bind at nanomolar concentrations to a receptor and block voltage-operated Ca2+ channels. In studies reported here, Harderian gland tissue homogenates from intact male hamsters exhibited significant dihydropyridine binding (Bmax = 1700 fmoles/mg protein) of high affinity (Kd = 1.1 nM). Tissue homogenates from female animals exhibited a similar Kd value (1.35 nM) but receptor density per mg protein was significantly reduced (Bmax = 270 fmoles). Dihydropyridine binding of Harderian gland tissue homogenates from castrated males was reduced greater than 80% (Bmax = 225 fmoles/mg protein). Treatment of castrated males with subcutaneous testosterone pellets resulted in significant restoration of dihydropyridine binding activity (approximately 80%, Bmax = 1630 fmoles/mg protein) with a comparable binding constant (Kd = 1.50 nM) as observed for noncastrated, control animals. Addition of testosterone (ex vivo) to homogenates from castrated hamsters did not restore dihydropyridine binding to control levels. These data indicate: (a) the Harderian gland from male hamsters exhibits significant dihydropyridine binding; (b) ligand binding is abolished following castration; and (c) significant restoration of dihydropyridine binding occurs following in vivo testosterone treatment. The dependence of dihydropyridine binding restoration upon in vivo steroid hormone administration suggests probable involvement of the steroid at the transcriptional level although non-genomic mechanisms such as the binding of testosterone to a receptor resident in the plasma membrane and subsequent activation of Ca2+ channels can not be ruled out.


Solitary calcium spike dependent on calmodulin and plasma membrane Ca2+ pump. Resealed human red cell ghosts were loaded with Fura-2, ATP, Mg2+, and either calmodulin (CaM) or, to prevent CaM activation of the Ca2+ pump, a synthetic peptide that antagonized endogenous CaM (an analogue of the CaM binding domain of protein kinase II, referred to as 'antiCaM'). The ghosts reduced the cytosolic concentration of ionized calcium ([Ca2+]i) to 193 +/- 60 nM (SD, n = 15) in a medium containing 1 mM Ca2+ and to 30 +/- 27 nM (SD, n = 62) in a medium without Ca2+ addition. Without ATP, i.e. no fuelling of the Ca2+ pump, the [Ca2+]i remained high (approx. 5 microM or higher). The simultaneous addition of the ionophore A23187 and Ca2+ rapidly increased the Ca2+ influx, which in the CaM loaded ghosts caused a solitary spike of [Ca2+]i, reaching maximum around 2 microM within 24 +/- 6 s (SD, n = 40). On the contrary, in the ghosts loaded with antiCaM, the addition of A23187 with Ca2+ raised [Ca2+]i during the first 2 min to a high level (2-4 microM) with no preceding spike. Pre-incubation of CaM-ghosts with Ca2+ diminished the height of the Ca2+ spike, and treatment with trypsin even removed the Ca2+ spike. The trypsin treatment activated the Ca2+ pump prior to the rise of [Ca2+]i, making the time-consuming CaM activation unnecessary. In conclusion, the Ca2+ spiking is dependent on a delayed CaM activation of the plasma membrane Ca2+ pump in response to a rapid increase of Ca2+ influx.


Beta-adrenergic effects on cellular Na, Mg, Ca, K and Cl in vascular smooth muscle: electron probe analysis of rabbit pulmonary artery. The effects of beta-adrenergic stimulation on the cellular content and subcellular distribution of Na, Mg, Ca, K and Cl were determined by electron probe X-ray microanalysis of muscles stimulated with 5-hydroxytryptamine. Isoproterenol caused a significant decrease in cytoplasmic and mitochondrial Na and Cl, and an increase in cytoplasmic Mg. Isoproterenol also significantly decreased total cytoplasmic Ca measured with small diameter probes, without affecting cellular Ca measured with large probes that included the sarcoplasmic reticulum (SR). The decrease in cytoplasmic Na and the effects on cytoplasmic and cellular Ca are consistent with, respectively, beta-adrenergic stimulation of the Na-pump and of Ca-uptake into the SR, but the beta-adrenergic increase in cytoplasmic Mg also raises the possibility of stimulated Na/Mg exchange.


Effect of 2H2O upon D-glucose metabolism in rat and human erythrocytes. When either rat or human erythrocytes were incubated for 90-180 min in bicarbonate-buffered media prepared in 2H2O rather than 1H2O, the generation of 3HOH from D-[5-3H]glucose, the production of either 14CO2 or 14C-labelled acidic metabolites from D-[U-14C]glucose and the production of L-lactic acid from unlabelled D-glucose were decreased, to a variable extent but, in no case, by more than 30%. Hence, total substitution of 1H2O by 2H2O provides a suitable tool to study by NMR the generation of 2H-enriched L-lactic acid generated from exogenous D-[1-13C]glucose or D-[6-13C]glucose and, hence, to further explore the diabetes-induced alteration of hydrogen isotopes intermolecular transfer in the reaction catalyzed by phosphoglucoisomerase.


Expression and methylation of the beta-subunit gene of prolyl 4-hydroxylase: in erythrocytes, tendon and cornea of chick embryos. It has recently been demonstrated that the beta-subunit of prolyl 4-hydroxylase (E.C. 1.14.11.2) is the same gene product as protein disulfide isomerase (PDI) and cellular thyroid hormone binding protein (THP). Therefore, it is very likely that the beta-subunit of the prolyl 4-hydroxylase gene serves as a house keeping gene in most cell types. In the present study, we examined the distribution of the chicken beta-subunit of prolyl 4-hydroxylase/protein disulfide isomerase (CPH beta/PDI) in erythrocytes, corneas and tendons of 13-, 17-, and 19-day-old chick embryos by immunohistochemistry using antibodies against CPH beta/PDI. Our data indicate that erythrocytes do not express the CPH beta/PDI gene whereas tendon cells express CPH beta/PDI at all developmental stages examined. The basal cells of corneal epithelium express CPH beta/PDI, but the superficial cell layers of stratified corneas of 19-day-old chick embryos do not. The expression of the CPH beta/PDI gene is also confirmed by in situ hybridization with cDNA encoding CPH beta/PDI. The results indicate that the expression of CPH beta/PDI in cornea is probably developmentally regulated. It has been suggested that methylation of genomic DNA is one of many possible regulatory mechanisms for gene expression. In order to examine whether methylation of genomic DNA may play any role in the expression of the beta-subunit gene, genomic DNA was isolated from corneas, tendons, and erythrocytes of individual 13-, 17-, and 19-day-old chick embryos. DNA samples were digested with Sma I and Eco RI, or Pst I and Sma I and followed by either Msp I, Hpa II, or Hha I and were then subjected to Southern hybridization with 32P-labeled genomic DNA fragments of CPH beta/PDI. Our results indicate that the CPH beta/PDI gene is methylated at the Hha I site in the 4th exon in erythrocytes whereas the same sites in tendon and cornea are hypomethylated. Examination of 5'-end flanking sequences of exon 1 of the CPH beta/PDI gene with the methylation sensitive endonucleases, Hha I and Hpa II did not reveal any difference in erythrocyte, cornea and tendon cells. Thus, our results indicated that DNA methylation may not play an important role in the expression of CPH beta/PDI.


Sample size determination under an exponential model in the presence of a confounder and type I censoring. In controlled clinical trials, random assignment of treatments to individuals is usually used to eliminate the effects of confounding variables. When there is censorship in data, however, confounding effects may not be automatically removed solely by random assignment of treatments to individuals under the exponential model. Therefore, it is important to incorporate the confounding effect into the sample size calculation even after randomization of treatments to individuals. In this paper, the discussion is restricted only to the situation where there are two comparison groups and one single Bernoulli confounding variable. Based on an exponential covariate model, an explicit sample size formula considering the confounding effect has been derived for the design of trials with type I censoring, in which an end time is fixed in advance and all responses occurring after that time are censored. The resulting sample size formula can also be applied to nonrandomized clinical trials. Finally, to provide insight into the influence of different factors on sample size calculation, a discussion on the effects of treatments, the confounder, the length of follow-up times for studied individuals, and the joint distribution of the treatment and the confounder has been included.


The future of statistics and statisticians in European regulatory affairs. Roles of statisticians in drug regulatory agencies include assessment of the adequacy of statistical aspects of the design of the programme of efficacy and safety studies, design of individual studies, data collection and quality assurance in each study, analysis of individual studies, interpretation of individual studies, and interpretation of results of the programme. These assessments should be made in collaboration with medical, pharmacological, and toxicological colleagues, and liaison with company statisticians before, during, and after submission of the application if the procedure is to be efficient. Recommendations for improving statistical quality of drug license applications and provision for their statistical review in Europe include: 1. Employment of more experienced, professional statistical assessors in both national and European Community (EC) regulatory authorities, with a wider group of supporting experts 2. Obligatory statistical coauthorship or validation of expert reports before submission of applications, and 3. Unified statistical guidelines for preparation of applications.


Enrollment in clinical trials: institutional factors affecting enrollment in the cardiac arrhythmia suppression trial (CAST). Recruitment and Enrollment Assessment in Clinical Trials (REACT), an NHLBI-sponsored substudy of the Cardiac Arrhythmia Suppression Trial (CAST), was conducted to assess factors associated with enrollment in clinical trials. We report on the relationships of institutional factors at CAST sites to patient enrollment. The proportion of CAST-eligible patients enrolling at each CAST site during the REACT study period was defined as the number of subjects enrolled divided by the sum of (1) the number enrolled plus (2) the number of eligibles who refused plus (3) the number of eligibles whose physicians refused to permit CAST personnel to attempt to enroll them. A questionnaire that included 78 questions regarding factors hypothesized to be associated with enrollment was completed between August 1988 and February 1990 by the nurse coordinators at all 112 CAST sites in the United States and Canada. Sixteen items were unanalyzable, and 37 of the remaining 62 were grouped into seven scales. The remaining items were analyzed individually. Enrollment proportions varied widely across the 112 CAST sites (mean 32.7% SD 22.6). Five variables or scales were included in the final multiple regression model (multiple R2 = .39). The most important of these was the proportion of eligible patients at a site cared for by medical staff other than private attending physicians (multiple R2 for this variable alone, .26). This proportion tended to be high in teaching hospitals. Other variables in this model that were associated with higher enrollment proportions included the number of days per week a nurse coordinator was present at the site, the number of nurse coordinator full-time equivalents at the site, fewer other clinical trials for which the nurse coordinator was responsible, and fewer perceived obstacles to enrollment. These findings indicate that enrollment was more successful at hospitals with higher proportions of eligible subjects cared for by fellows, housestaff, and service attending physicians and at institutions with the committed presence of a nurse-coordinator.


Single vs. double data entry in CAST.

Single vs. double data entry in CAST. An experiment was conducted to determine whether, using microcomputer-based data entry, double data entry (DE) significantly lowers data entry keying error rates when compared to single entry (SE). Clinical centers of the Cardiac Arrhythmia Suppression Trial (CAST) participated in a randomized crossover design experiment comparing SE and DE. A total of 42,278 data items (fields) were checked for consistency between the paper data form and the computer database. The overall error rate was 19 per 10,000 fields. Error rates were 22 and 15 per 10,000 fields for SE and DE, respectively; P = .09 by Poisson regression. DE took 37% longer than SE, costing each clinic approximately an extra 90 min per month.


Pravastatin, lipids, and atherosclerosis in the carotid arteries: design features of a clinical trial with carotid atherosclerosis outcome. The Pravastatin, Lipids, and Atherosclerosis in the Carotids trial (PLAC-II) was initiated in 1987 and is the first double-masked randomized clinical trial with progression of early extracranial carotid atherosclerosis as an outcome variable. The trial will compare a lipid-lowering agent (pravastatin, a hydroxymethylglutaryl CoA reductase inhibitor) with placebo for ability to retard the rate of progression of extracranial carotid atherosclerosis over 3 years. Inclusion criteria consisted of prevalent coronary artery disease, moderately elevated low-density lipoprotein (LDL) cholesterol (between the 60th and 90th percentiles), and the presence of at least one extracranial carotid artery atherosclerotic plaque that had an intimal-medial thickness (IMT) > or = 1.3 mm as visualized by B-mode ultrasound. Of approximately 650 patients who qualified on the basis of coronary disease and elevated LDL cholesterol, 55% were excluded because of B-mode criteria. One hundred and fifty-one males and females 50-75 years of age were recruited. Random allocation produced placebo-treated and test-treated groups that were similar for baseline historical data, physical findings, laboratory tests, lipid values, and B-mode characteristics. Baseline concentrations of plasma total cholesterol, LDL cholesterol, and high-density lipoprotein (HDL) cholesterol were 234, 166, and 41 mg/dl, respectively. Baseline plasma concentration of triglyceride was 170 mg/dl. Despite selection of participants whose arteries, overall, were suitable for the trial, individual segments in some participants could not be visualized. Ninety-seven percent of the individual carotid artery segments were visualized in the common carotid, 88% in the bifurcation, and 63% in the internal carotid artery. Far walls were slightly more often visualized than near walls, and nonvisualization was most common for the near wall of the internal carotid. Nonvisualized segments were comparable between both treatment groups. The distribution of arterial walls with qualifying plaque of > or = 1.3 mm IMT was similar for the two groups, and the two groups were also comparable for the primary outcome determinant, mean maximum IMT (mean of maximum of all visualizable sites, 1.32 mm for each treatment group). There are special problems related to recruitment and evaluation of patients for a clinical trial such as this, but the atherosclerosis outcome measurement markedly enhances power and compensates for difficulty in recruitment.


Fractal properties of herpes simplex dendritic keratitis. Fractal geometry can be used to analyze the complexity and self-similarity of many natural forms and structures. By using three techniques for fractal dimension estimation, a group of 11 herpes simplex virus dendritic corneal ulcers was analyzed and found to possess outlines with fractal properties. The mean surface fractal dimension approached 1.4 over 1.5 decades of scale. The demonstration and quantification of the fractal dimension of dendritic lesions is important in the understanding of their behaviour and, at a more fundamental level, the planar spread of viral infection.


Pretreatment with topical 0.1% (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine inhibits adenovirus type 5 replication in the New Zealand rabbit ocular model. Currently there is no clinically effective antiviral agent for the prevention or treatment of ocular adenoviral infections. Using a paired-eye, masked design, we tested the antiviral efficacy of topical 0.1% (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine in the New Zealand rabbit ocular model after topical and intrastromal inoculation with 100 microliters (4 x 10(5) plaque-forming units per eye) of adenovirus type 5 McEwen, a clinical isolate. Prevention studies involved pretreatment (six times a day) 1 day before inoculation and continuing for 4 additional days. Compared with the control eyes, the pretreated eyes showed a significant reduction in the peak viral eye titers on days 3, 4, 5, and 7 after treatment (p < 0.03-0.005), and a reduction in the duration of viral sheeding (p < 0.02). Rebound increase in adenoviral titers was detected in five of 20 eyes (25%) after cessation of treatment, suggesting a therapeutic effect and a need for further studies to optimize the treatment regimen.


Relationship between absorption of radiolabeled soluble insulin, subcutaneous blood flow, and anthropometry. To evaluate the interrelationships between the rate of absorption of soluble insulin, SCBF, and anthropometry in normal subjects. In 12 normal men (age range 23-30 yr, BMI 18.2-41.3 kg/m2), simultaneous assessment of the absorption of 125I-labeled soluble insulin and SCBF (99mTc clearance) was performed, on separate study days, for the anterior abdominal wall, anterior midthigh, and the upper arm sites. Each site was examined in a randomized order on two separate occasions. Absorption of 125I-soluble insulin was determined by external monitoring of residual radioactivity levels at the injection site for 6 h postinjection. Residual radioactivity level-time curves, including the characteristic early phase of slow absorption of soluble insulin (the lag phase), were described using two- and three-parameter biexponential models. Anthropometric measurements included BMI, ultrasonic measurement of the subcutaneous adipose tissue layer, and caliper skin fold thickness at the anterior abdominal wall, biceps, triceps, anterior midthigh, and subscapular sites. A highly significant positive relationship was observed between the rate of absorption of 125I-soluble insulin and SCBF (rS = 0.44-0.52; P < 0.01-0.001). The duration of the lag phase was inversely correlated with SCBF (rS = 0.34 - 0.51; P < 0.01-0.001). Inverse relationships also were observed for the subjects' degree of adiposity with the rate of soluble insulin absorption (rS = -0.43(-)-0.71; P < 0.001) and SCBF (rS = -0.27(-)-0.62; P < 0.05-0.001). Significantly shorter lag phase was observed for the abdominal site compared with thigh and arm injection sites (P < 0.05-0.01). The rate of absorption of soluble insulin, including during the lag phase, is positively correlated with SCBF. Increasing adiposity prolongs the duration of the early lag phase and reduces the rate of absorption of soluble insulin and SCBF.


Comparative features of comedo and noncomedo ductal carcinoma in situ of the breast on fine-needle aspiration biopsy. To determine whether fine-needle aspiration biopsy (FNAB) can differentiate between comedo (C-DCIS) and noncomedo ductal carcinoma in situ (NC-DCIS), we reviewed retrospectively the preoperative FNAB and surgical biopsy slides of 13 cases of DCIS with adequate cytologic material. Eight were NC-DCIS and 5 were C-DCIS. Three (60 percent) of the C-DCIS and 7 (88%) of the NC-DCIS were nonpalpable lesions biopsied under conventional mammographic guidance. Three (60%) of the C-DCIS but only 2 (25%) of the NC-DCIS were considered either suspicious or positive for malignancy on FNAB, the remainder in both groups being atypical. A statistically significant difference in marked nuclear pleomorphism (60% of C-DCIS vs. 0% of NC-DCIS, P = 0.04) and large nucleoli (60% of C-DCIS vs. 0% of NC-DCIS, P = 0.04) was observed between these 2 groups. DCIS is morphologically diverse, and it appears that the cytologic features of individual cells on FNAB may distinguish C-DCIS from NC-DCIS.


Metastatic hepatocellular carcinoma of the breast, simulating gynecomastia: diagnosis by fine-needle aspiration biopsy. Hepatocellular carcinoma (HCC) may uncommonly present with distant metastasis in the absence of a documented neoplasm in the liver. The authors herein describe the case of a 60-year-old man with cirrhosis who developed unilateral enlargement of the breast and a subareolar mass. This problem was clinically thought to represent gynecomastia, but a mammary fine-needle aspiration biopsy demonstrated a malignant epithelial neoplasm composed of large granular amphophilic cells. Bile pigment was visualized in the tumor on aspirate smears and cell block preparations; immunostains showed reactivity for cytokeratin and alpha-fetoprotein, but there was no positivity for epithelial membrane antigen, gross cystic disease fluid protein-15, vimentin, estrogen receptors, progesterone receptors, or S100 protein. These results indicated a diagnosis of metastatic HCC, which was subsequently confirmed by computed tomography of the abdomen.


Cystic nephroma: cytologic findings in fine-needle aspiration cytology. This report presents the fine-needle aspiration cytology (FNAC) findings of a multicystic renal tumor found in a 3-year-old child. The smears contained benign epithelial cells isolated or arranged in sheets of uniform cells strongly suggesting the lining of the cysts. The combination of the imaging data with the FNAC findings favoured the diagnosis of cystic nephroma (CN), a benign renal tumor that is cured by surgery. Surgical pathology confirmed the diagnosis. CN should be added to the list of tumors of the kidney in infancy that appear to be diagnosable by FNAC/biopsy.


Interaction of rat peritoneal macrophages with homologous sialidase-treated thrombocytes in vitro: biochemical and morphological studies. Detection of N-(O-acetyl)glycoloylneuraminic acid. Sialidase treatment of rat thrombocytes led to an increased binding of these cells to homologous peritoneal macrophages, but had no significant effect on the rate of phagocytosis during the experimental time. As revealed by electron microscopy, the partially desialylated thrombocytes adhere to macrophages predominantly via a small part of the membrane in a way that the discoidal cells adopt a vertical position with regard to the macrophage surface. One adherent macrophage was able to bind up to 55 sialidase-treated thrombocytes. Maximum binding was already reached after release of 13% of sialic acids. This interaction could be inhibited by free D-galactose and compounds with terminal D-galactose residues. Bound thrombocytes were released from the macrophages by treatment with lactose or EDTA. These experiments suggest that the interaction is mediated by a galactose-specific receptor on the macrophage surface and that galactose on thrombocytes is not recognized if it is masked by terminal sialic acid residues. The total sialic acid amount of the thrombocytes studied was about 70 micrograms sialic acid/10(10) cells being composed of 78% N-glycoloylneuraminic acid, 17% N-acetylneuraminic acid and 5% of the novel sialic acid N-(O-acetyl)glycoloylneuraminic acid, which was identified by mass spectrometry. Sixty-two percent of these sialic acids were susceptible to enzymic hydrolysis with Vibrio cholerae sialidase.


Transcriptional and posttranscriptional control of connexin mRNAs in periportal and pericentral rat hepatocytes. Distinct patterns of expression of gap junction, or connexin, mRNAs were observed in periportal vs. pericentral hepatocytes. The two cellular fractions (isolated from rat livers by perfusion) were more than 90% parenchymal, as determined by flow cytometry for a hepatocyte-specific marker. The periportal and pericentral fractions were identifiable due to enrichment in enzymatic activities previously shown to be differentially expressed in the respective regions of liver. Northern blot analyses revealed that mRNA encoding connexin 26 was 2.8 times more abundant in the periportal than in the pericentral cells, while connexin 32 mRNA was equally distributed. Messenger RNA from each fraction was radiolabeled in order to compare the relative abundance of the connexin mRNAs in each fraction. The ratio of connexin 26 to connexin 32 mRNA in the portal fraction was about 0.085, and in the central fraction about 0.038. Connexin 26 mRNA was transcribed, however, at a faster rate than connexin 32 mRNA by nuclei isolated from both cellular fractions. Connexin 26 mRNA was transcribed at 3.9 times the rate in nuclei from the periportal than from the pericentral cells. These data suggest that while the zonation of connexin 26 mRNA synthesis in liver appears to be controlled transcriptionally, posttranscriptional regulatory mechanisms determine the relative abundance of the connexin mRNAs.


Epstein-Barr virus infection and associated diseases in children. II. Diagnostic and therapeutic strategies. Epstein-Barr virus (EBV), an ubiquitous human B lymphotropic virus, is the cause of infectious mononucleosis. Moreover, EBV infection can be followed by lymphoproliferative diseases in patients with inherited and acquired immunodeficiencies. Primary EBV infection may be a threat to all children after marrow or organ transplantation or those receiving chronic immunosuppressive treatment for various other reasons. The virus has been also implicated in the pathogenesis of different malignant tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin disease and also some T-cell lymphomas. This review focuses on various aspects of virus-host interactions, immune mechanisms of the host, and the still experimental therapeutic approaches in EBV-associated diseases.


Influence of (+/-)-CP-96,345 and SR 48968 on electrical field stimulation of the isolated guinea-pig main bronchus. The aim of this study was to investigate the effects of (+/-)-CP-96,345 and SR 48968, two new nonpeptide antagonists of neurokinin NK1 and NK2 receptors, respectively, on the response of isolated guinea pig main bronchi to electrical field stimulation (EFS). Bronchi were stimulated transmurally with biphasic pulses (16 Hz, 1 ms, 320 mA for 10 s) in the presence of indomethacin (10(-6) M) and propranolol (10(-6) M). Two successive contractile responses were observed. Both responses were abolished by tetrodotoxin (10(-6) M) whereas only the first rapid phase was abolished by atropine (10(-6) M). The late and prolonged second phase was strongly reduced by the neurokinin A (NK2) receptor antagonist SR 48968 (10(-11) to 10(-8) M) with an EC50 of 0.056 nM and a maximal inhibition of 83.3 +/- 10.8% (10(-8) M, n = 4). This second response was partially inhibited by the substance P (NK1) receptors antagonist (+/-)-CP-96,345 (10(-8) to 10(-6) M). An incubation of 2 h was necessary for SR 48968 to inhibit the EFS-evoked noncholinergic contraction. These results confirm that EFS of guinea-pig bronchi involves stimulation of cholinergic and noncholinergic excitatory nerves and demonstrate that the new developed tachykinin receptors nonpeptide antagonists (+/-)-CP-96,345 and especially SR 48968 are potent inhibitors of the noncholinergic contraction induced by EFS of the isolated guinea-pig main bronchus.


Cellular kinetics of inflammation in the pleural space of mice in response to the injection of exogenous particles. CD-1 mice were used to study the cellular kinetics of the inflammatory response of the pleural space to the injection of 250 micrograms of silica or of tungsten microparticles. The pleural exudates were collected by lavage of the serous cavity of mice that were sacrificed at 30 min and up to 7 days after the intrapleural instillation of the particles. The samples were studied by light and electron microscopy (transmission and scanning modes); the quantitative cellular kinetics of the inflammation was determined by leukocyte counting in exudates using cytocentrifuge preparations. The normal resident population of cells of CD-1 mice was made up of (2.47 +/- 0.37) x 10(6) cells. It consisted mostly of macrophage-like cells ((2.03 +/- 0.26) x 10(6) cells, 82% of total cells), some lymphocytes ((0.37 +/- 0.07) x 10(6) cells, 15% of total cells), a few mast cells and eosinophilic granulocytes (1-2% of total cells). The initial inflammatory reaction (30-60 min after injection) was characterized by a decrease in the number of cells harvested from the pleural space. This was followed by an intense recruitment of granulocytes and monocytes that resulted in a peak of intrapleural cells at 24 h ((16.8 +/- 4.0) x 10(6) cells induced by silica particles and (18.3 +/- 4.2) x 10(6) cells induced by tungsten particles). In tungsten-injected mice (but not in silica-treated animals) the enhancement in the number of intrapleural macrophages continued up to 72 h after particle injection. The highest percentage of macrophages with ingested tungsten (50% of total macrophages) was found early (6 h) and decreased thereafter; at day 7 it encompassed just 17% of the macrophages. Injection of any of the two particulates led to the disappearance of mast cells from the pleural space of mice. Silica particles attracted a high number of eosinophils to the pleural cavity of mice. Light and electron microscopy documented that pleural macrophages underwent striking morphological changes during the inflammatory response: the phagocytes showed marked increase in size and in number of surface processes, and their cytoplasm often contained large amounts of the injected particles and also of cellular debris. This study establishes the mouse as a reliable animal model to study the dynamics of the pleural space and it offers a precise definition of the cellular kinetics of inflammation in this serous cavity. The data indicate that the kinetics of experimental pleural inflammation induced by particulates may depend on the nature of the injected particles.


Opioid receptor mediation of the hypothermic response to caffeine. Caffeine and other methylxanthines induce a dose-dependent reduction in core body temperature in mice. These experiments investigated the effects of neurotransmitter and neuromodulator antagonists on caffeine-induced hypothermia. Pretreatment with the alpha 2-adrenoceptor antagonist, atipamezole; the beta-adrenoceptor antagonist, propranolol; the dopamine antagonist, haloperidol; or the benzodiazepine receptor antagonist, flumazenil had no intrinsic effects on core body temperature nor did they interact significantly with the hypothermic effects of caffeine. The alpha 1-adrenoceptor antagonist, prazosin and the 5-HT receptor antagonist, metergoline significantly enhanced the hypothermic effects of caffeine, probably involving a combined effect with their intrinsic hypothermic actions. Pretreatment with the opiate receptor antagonist, naloxone (3 mg/kg i.p.), had no intrinsic effect on core body temperature but attenuated the hypothermic effect of caffeine reflected in a parallel shift to the right in the caffeine dose-effect curve. The naloxone-induced attenuation of the hypothermic effects of caffeine was also seen to be dose-dependent. The results reveal that opiate receptors (but not adrenoceptors, 5-HT, dopamine or benzodiazepine receptors) may play a role in modulating the hypothermic action of caffeine and possibly other methylxanthines.


Discovery of a potent atrial natriuretic peptide antagonist for ANPA receptors in the human neuroblastoma NB-OK-1 cell line. The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).


The distribution of the DHFR genes in trimethoprim-resistant urinary tract isolates from Taiwan. Between July 1987 and June 1989, 1054 urinary isolates of enterobacteria from Kaohsiung, Taiwan were studied for their trimethoprim resistance. Trimethoprim resistance was defined as MIC greater than 4 micrograms/ml and high-level resistance by MIC greater than 1000 micrograms/ml. The incidence of trimethoprim resistance increased from 33.6% in 1987 to 42.1% in 1989. Among the resistant strains studied, 90% were resistant to high levels of trimethoprim. An increase in the proportion of resistant strains (33.9-46.3%) exhibiting high-level non-transferable trimethoprim resistance was noted. The distribution of the dihydrofolate reductase (DHFR) genes by colony hybridization in 374 trimethoprim-resistant isolates revealed the presence of type I and type V DHFR genes in most of these isolates (45.4% and 10.4% respectively). Type I was predominant in Escherichia coli whereas type V was frequently seen in Enterobacter spp. None showed homology with the type II and type III DHFR probe DNA. In addition, transposon Tn7 was present in 7.8% of 374 trimethoprim-resistant enterobacteria.


Genetic relationships between southern African SAT-2 isolates of foot-and-mouth-disease virus. Sequencing of part of the 1D gene of foot-and-mouth disease virus was used to determine the relationships between SAT-2 viruses isolated from outbreaks which occurred in cattle in Zimbabwe and Namibia and in impala in South Africa between 1979 and 1989. The results demonstrated that the outbreaks in different countries were unrelated. Surprisingly close relationships were shown between all SAT-2 viruses isolated from cattle in Zimbabwe since 1983 but the two major epizootics which occurred in 1989 were caused by viruses which were clearly different. Conversely, two apparently unrelated outbreaks in impala in South Africa were caused by viruses which could not be distinguished.


Platelet receptor expression on three human megakaryoblast-like cell lines. Platelets, the progeny of bone marrow megakaryocytes, are nonnucleated cells; many platelet proteins, including platelet membrane receptors, are believed to be derived from megakaryocytes. Several hematopoietic cell lines that exhibit megakaryocytic characteristics have been established as models for the study of megakaryocyte biology. We report here the screening of platelet receptor expression, in terms of functional coupling with the formation of two second messengers, calcium and cAMP, in three cell lines exhibiting megakaryoblastic properties: HEL, MEG-01, and DAMI. We show that all these cell lines respond to thrombin, ADP, epinephrine, and prostaglandin E1 (PGE1). However, transmembrane signaling pathways appear partly different from those present in mature platelets, because the action of thrombin was found to be positively coupled with the cAMP pathway, in addition to that of calcium, and because PGE1, which interacts with the cAMP pathway, also raises intracellular calcium levels in the three cell lines studied. Furthermore, an endothelin-1-induced increase in intracellular calcium level was observed in MEG-01 cells, strongly suggesting the expression of endothelin receptors on platelet precursors cells, whereas the presence of such receptors is controversial on platelets. These cell lines should prove useful in further studies of the expression and molecular pharmacology of platelet receptors on platelet precursor cells, as well as for the investigation of functional roles for platelet receptors on megakaryoblastic cells.


Receptor mediated mineralocorticoid action in alga cell mutants. The multiplication of Chlamydomonas cells can be arrested by the spirolactone derivative RU 26752 and this is fully reversible by the natural hormone aldosterone. Continuous growth in the presence of RU 26752 led to the isolation of a population subsequently resistant to the action of mineralocortoid analogues, due possibly to the selection of mutant cells. Immunophotochemical evidence is provided for a 52 kDa protein that possesses functional steroid and DNA binding domains. Alga cells therefore appear to respond to steroid hormones in a manner similar to the mammalian systems, possibly via a receptor that may represent a pygmy ancestor of the latter day steroid receptor superfamily.


Phosphorylation of smg p21B in rat peritoneal mast cells in association with histamine release inhibition by dibutyryl-cAMP. IP3 formation and histamine release from rat peritoneal mast cells stimulated by compound 48/80 were dose-dependently inhibited by Bt2cAMP. These inhibitions were restored to the control level in the presence of H-8, a protein kinase A inhibitor. The 22 kDa protein in mast cells was revealed as a markedly phosphorylated protein by incubating with Bt2cAMP, and this phosphorylation was also diminished by H-8. The 22 kDa phosphoprotein of rat mast cells comigrated with phosphorylated smg p21B, purified from human platelets and phosphorylated by protein kinase A in cell-free system, in both one- and two-dimensional PAGE analysis. Moreover, 22 kDa protein in mast cells was identified as smg p21B by immunoblot analysis using an antibody against smg p21B. From the present study, it became clear that smg p21B is phosphorylated by means of protein kinase A system in rat peritoneal mast cells, and it was assumed that phosphorylated smg p21B plays some important role in the suppression of IP3 formation and histamine release from rat peritoneal mast cells.


Two subforms of eukaryotic topoisomerase I. Purification and structure-function relationships. A new method for isolation of eukaryotic topoisomerase I from calf thymus and from Jurkat-1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes.


Tumor necrosis factor alpha stimulates sphingomyelinase through the 55 kDa receptor in HL-60 cells. Tumor necrosis factor alpha (TNF alpha) stimulated rapid (seconds) hydrolysis of sphingomyelin in HL-60 cells, formation of phosphocholine (PCho) and a decrease in choline. The response to TNF alpha was concentration dependent with a maximal effect at 3-10 nM. The monoclonal antibody (mAb), htr-9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in intact cells. In contrast, the mAb, utr-1, which behaves as an antagonist at the 75 kDa receptor subtype, had no effect on sphingomyelin hydrolysis either on its own or in the presence of TNF alpha. In addition, htr-9 or TNF alpha stimulated hydrolysis of sphingomyelin in a membrane fraction of HL-60 cells. These results are consistent with a role of sphingomyelin hydrolysis as an early event in the signalling mechanism of TNF alpha, and suggest that this pathway is activated through the 55 kDa subtype of the TNF receptor.


Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase. Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.


Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. The Alzheimer-like state of tau protein includes phosphorylation by a proline-directed Ser/Thr kinase present in normal or pathological human brain. Extending earlier results on MAP kinase, we show here that the proline-directed kinase, GSK3, can induce an Alzheimer-like immune response involving several distinct and phosphorylatable epitopes at Ser-Pro motifs, as well as a gel mobility shift, similar to MAP kinase. Both kinases behave like microtubule-associated proteins in that they co-purify through cycles of assembly and disassembly, and both kinases are directly associated with paired helical filaments.


Augmentation of retinoic acid-induced granulocytic differentiation in HL-60 leukemia cells by serine/threonine protein phosphatase inhibitors. To evaluate the involvement of protein phosphatases (PP) in differentiation of human myelogenous leukemia HL-60 cells, we made use of potent inhibitors of PP1 and PP2A, calyculin-A (CAL-A) and okadaic acid (OKA). CAL-A and OKA could augment all-trans retinoic acid (ATRA)-induced granulocytic differentiation, whereas the differentiation toward macrophage lineage by 12-o-tetradecanoylphorbol acetate (TPA) was unchanged in the presence of CAL-A. CAL-A augmented the phosphorylation of 18K, 23K and 30K proteins induced by ATRA. The PP1 and PP2A were identified and were present mainly in the cytosol of HL-60 cells. These results suggest that either PP1 or PP2A or both may be involved in regulating granulocytic differentiation of HL-60 cells.


Molecular basis for the insensitivity of the Monarch (Danaus plexippus) to cardiac glycosides. The Monarch (Danaus plexippus) sequesters cardiac glycosides for its chemical defence against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera, such as Manduca sexta and Creatonotos transiens are sensitive and intoxicated by ouabain. Ouabain inhibits the Na+,K(+)-ATPase by binding to its alpha-subunit. We have amplified and cloned the DNA sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain-sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of about 12 amino acids. This change may explain the ouabain insensitivity.


[The role of dermorfin in regulating hibernation processes in mammals]. Administration of dermorphin (0.1 mg/kg, subcutaneously) induced a drop of body temperature, deceleration of initial vegetative parameters and development of hibernation-like state in susliks. The delayed effects involved a disorder in conditioning. The immunisation of hibernating susliks with the dermorphin conjugate leads to a gradual waking up of the animals, normalising of conditioning in all the parameters and occurrence of a motor component in avoidance conditioning. A possible specific role of the dermorphin in the mechanisms of hibernation, is discussed.


[The action of high temperature on beta-adrenoreceptor activity and catecholamine synthesis]. Hyperthermia and overheating of the rat isolated organs induce significant changes of the binding of receptors' specific blockers and catecholamine synthesis. One of the main mechanisms of the organism response to the temperature actions involves a local rearrangement in intensity of different stages of the adrenergic process. Possible significance of these changes in the hypothalamus for synchronisation of the organism's response to extreme temperature effects, is discussed.


[The zona reticularis and the regulation of its activity under stress exposures]. A single stress effect activated the fascicular zone whereas repeated stress effects activated both the fascicular and the reticular zones in male rats. Therefore, these zones seem to have different modes of regulation despite the fact that their common basic regulating agent is ACTH. A new scheme of regulation of the reticular zone in repeated stress effects, has been presented.


[The function of the hypothalamo-hypophyseal-adrenal system in the progeny of parathyroidectomized rat dams]. Parathyroidectomy in rats was found to lower the calcium level in the litter's blood plasma, activate the hypothalamo-hypophyseal-adrenal system post partum and reduce it in ontogenesis. The system's development and postnatal activity seems to depend on the homeostasis of calcium.


[The effect of 6-hydroxydopamine injections into the nucleus accumbens and substantia nigra on rat behavior]. Bilateral infusion of 6-hydroxydophamine (15 mg/ml) into the n. accumbens and substantia nigra accelerated the avoidance conditioning and increased the dopamine and 3,4-dihydroxiphenylacetic acid levels in the striatum as compared with the rats which had only been injected the same agent in the substantia nigra alone. Infusion of this agent into the n. accumbens can be used to compensate for the nigrostriatal deficit.


[The action of amtizol on the plastic properties of the membrane of Retzius' neuron in the leech]. Extracellular application of amtysol decreased the amplitude and increased the duration of evoked APs, not affecting the form of spontaneous APs in the leech Retzius' neurons. Besides, a disorder in the habituation process occurred which only normally develops during a high-frequency synaptic activation of the neurons.


[The vagotropic action of peptides isolated from the brain of hibernating susliks]. Dipeptide ASP-TYR extracted from the brain of hibernating squirrels was found to potentiate vagal effect on the cardiac rhythm, whereas pentapeptide neokyotorphin--to diminish it. In both cases the effect was due to an alteration of the tonic component's strength in the vagal chronotropic effect. The selective peptidergic modulation of the vagal effect on the cardiac rhythm suggests different physiological roles of the tonic and synchronizing components in regulation of the heart.