Analysis and examination of cytokine interactions by the median-effect model: an example with antiviral action of tumor necrosis factor and interferon-gamma. A mathematical model based on the median effect principle was used to demonstrate its usefulness in studying cytokine interactions. The inhibition of cytopathological effects due to encephalomyocarditis virus (EMCV) by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF) in WISH cells using a microtiter assay was employed. The model was used to analyze the characteristics of the dose-effect relationships of IFN-gamma, TNF, and their combination. The method also was used to define synergism as opposed to additive interaction. This application of the median-effect model proved to be easy and accurate and could be useful in the study of interactions in cytokine research.


Mucus-producing adenopapillary carcinoma of the oral cavity. Mucus-producing adenopapillary carcinoma is a rare neoplasm of the oral cavity. The literature is reviewed and a case described, with histological and ultrastructural findings of a tumour which involved the upper lip and recurred following local excision.


Brachial plexus injury: a survey of incidence and referral pattern. The aim of this study was to establish the incidence and the pattern of referral of injuries to the brachial plexus for a single calendar year: 1987. A questionnaire was sent to every orthopaedic consultant and referral centre in the United Kingdom. Replies were received from 402 surgeons. It is estimated that in 1987 there were at least 336 patients with traumatic injuries and 137 patients with other problems related to the plexus. Only 145 patients were seen at a referral centre. The anatomical nature, mode of injury and methods of treatment were analysed for both the referred and unreferred patients. We found that injuries to the brachial plexus are far more common than is generally appreciated, but only a minority of patients are referred for specialist evaluation and treatment.


Mobilizable intracellular pool of p55 (type I) tumor necrosis factor receptors in human neutrophils. The distribution of type I (p55) and type II (p75) tumor necrosis factor receptors (TNF-Rs) in human polymorphonuclear neutrophils (PMNs) was analyzed by Western blotting of subcellular fractions obtained by centrifugation of PMN cavitates on Percoll density gradients. In resting PMNs, the p55 receptor was associated with both gamma and beta fractions, enriched in plasma membranes and specific granules, respectively, whereas the p75 TNF-R was located only in the gamma fraction. Intracellular p55 TNF-R bound 125I-labeled TNF in a ligand blot assay and migrated as a diffuse band of 46-60 kd, similar to the plasma membrane receptor. Activation of PMNs with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), in conditions that induced the selective release of the tertiary granule marker gelatinase, caused the shedding of most of membrane p55 TNF-R as a 28-kd soluble form but had little effect on the distribution of the receptors in beta fractions. By contrast, when specific granule secretion was induced in the presence of cytochalasin B, a marked decrease in reactivity of beta fractions with anti-p55 TNF-R antibody was observed. At the same time, the amount of soluble 28-kd fragment found in the supernatant was increased. Thus, the intracellular pool of p55 TNF-R is associated with functional secretory granules and is redistributed in response to PMN activation.


Postreceptor events associated with human neutrophil activation by interleukin-8. Recombinant human monocyte-derived interleukin-8 (IL-8M), recombinant human endothelium-derived IL-8 (IL-8E), and a recombinant human truncated form of IL-8 (IL-8T) stimulated a time-dependent (t 1/2 approximately 2-3 s) and concentration-dependent (0.1-100 nM) release of azurophil (myeloperoxidase) and specific (vitamin B12 binding protein, gelatinase) granule constituents from cytochalasin B-treated human neutrophils (HNs) wherein IL-8T = IL-8M greater than IL-8E. An increase in the cytosolic free calcium concentration ([Ca2+]i) was greater in IL-8T- than in IL-8M- or IL-8E-activated HNs, and IL-8T was more potent than either IL-8M or IL-8E in sequentially desensitizing the HNs to the effects of the other IL-8 forms. IL-8 induced a time- and concentration-dependent (0.1-100 nM) increase in the production of inositol 1,4,5-trisphosphate (IP3) in HNs. U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), a potent inhibitor of phospholipase C-catalyzed events in HNs, suppressed IL-8-triggered IP3 production, increased [Ca2+]i and granule exocytosis in HNs. The membrane-associated activity of the alpha and beta subtypes of protein kinase C was significantly enhanced in IL-8-activated cells.


Hepatitis C in chronic liver disease: an epidemiological study based on 566 consecutive patients undergoing liver biopsy during a 10-year period. We analysed the presence of hepatitis C virus (HCV) antibodies in 566 patients undergoing liver biopsy. While over 20% of the patients were anti-HCV positive according to ELISA, only 13.8% had HCV antibodies when tested with a four-antigen recombinant immunoblot assay (RIBA 2). At the time of inclusion in the study, most patients were asymptomatic, irrespective of whether they were HCV-positive. Histological findings in anti-HCV-positive patients were chronic persistent hepatitis, chronic active hepatitis or cirrhosis in greater than 75% of cases. Only four of the patients who were anti-HCV-positive according to the RIBA 2 had autoimmune chronic active hepatitis. Risk behaviour could be identified in the majority of cases. Community-acquired sporadic cases were rare (12%). Of the 153 patients who died during follow-up, 23 subjects were anti-HCV positive. Although age- and sex-adjusted survival was not shorter in anti-HCV-positive patients than in anti-HCV-negatives, the risk of hepatocellular cancer was higher (P = 0.01). We conclude that HCV infection is associated with chronic liver disease, even when critical evidence of viral aetiology is slight. Truly sporadic cases are rare. Patients infected with HCV are at increased risk of developing hepatocellular cancer.


Ultimobranchial body and parathyroid glands of the freshwater snake Natrix piscator in response to vitamin D3 administration. Vitamin D3 (20 I.U./100 g body wt) was administered to the freshwater snake Natrix piscator for 15 days. Elevation of serum calcium and inorganic phosphate levels was observed after the treatment. The ultimobranchial body became active whereas the parathyroid glands exhibited reduced activity.


Role of thyroid hormone in intermediary metabolism of propranolol or alloxan-treated Calotes versicolor. Administration of L-thyroxine (T4) to thyroidectomized Calotes versicolor significantly increased the activity of glucose-6-phosphatase (G-6-Pase) (liver and kidney), the concentrations of blood glucose and total protein (liver and kidney), and decreased hepatic cholesterol when compared to thyroidectomized lizards. Propranolol injections in thyroidectomized lizards increased the cholesterol concentration and did not change the other parameters. The activity of G-6-Pase and blood glucose content was stimulated, whereas the total protein and cholesterol contents were decreased after alloxan treatment. Administration of T4 to thyroidectomized animals pretreated with propranolol or alloxan significantly elevated the activity of G-6-Pase, the concentrations of blood glucose, and total protein, and reduced hepatic cholesterol level when compared to drug-treated lizards. From the results, it is evident that thyroid hormone has an independent stimulatory influence on intermediary metabolism in C. versicolor irrespective of the involvement of adrenaline or insulin.


Chromosome assignments of the human TNF p55 and p75 receptor genes. At least two different receptor molecules have been described that are capable of binding tumor necrosis factor alpha, a cytokine that plays an important role in inflammation and antitumor activity. Comparative analyses at the nucleotide sequence level suggest that these receptors are members of a newly defined protein family that also includes human and rat nerve growth factor receptors. In this study, we determine the chromosome assignments of the human TNF alpha receptor genes, one of which may have evolved as part of a conserved Hox locus-containing chromosome segment.


Atrial natriuretic peptide clearance receptors in trout: effects of receptor inhibition in vivo. Inactivation of circulating atrial natriuretic peptides (ANP) by specialized clearance (C) receptors has been characterized in mammals but has not been examined in fish. In the present study arterial blood pressure, urine flow, and urine electrolytes were measured in chronically cannulated rainbow trout, Oncorhynchus mykiss, during infusion of the specific C receptor inhibitor, SC-46542. C receptor inhibition decreased blood pressure and pulse pressure, increased heart rate and urine flow, but did not affect urinary electrolyte concentrations. These responses are consistent with those produced by exogenous ANP administration and indicate that: (1) trout possess C-type receptors capable of ANP inactivation, and (2) ANP-like molecules are continuously released and metabolized by trout in vivo. Phosphoramidon, an inhibitor of neutral endopeptidase, did not enhance the SC-46542 response, indicating that C receptors predominate in ANP inactivation in these fish.


Fiber and weight management.

Fiber and weight management. Fiber is the portion of plant cells not digested in the human small intestine. Benefits of fiber consumption have been documented in treatment of obesity and obesity-related risk factors. In the last century carbohydrate intake and, thus, fiber intake have declined in the U.S. diet at the same time obesity has increased. Higher fiber intake can be achieved by adding high fiber foods or supplements to the diet. A combination of water soluble and water insoluble fiber sources offer the greatest health and weight control benefits. A high fiber weight-reducing diet is most beneficial as part of a lifestyle modification program for weight management.


Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder). Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.


Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes. Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS)


Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers. Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.


Acute sensory and autonomic neuropathy: possible association with coxsackie B virus infection. This report describes a 26 year old woman with a Coxsackie B virus infection complicated by an acute pandysautonomic and sensory neuropathy. Electrophysiological studies suggested an axonal neuropathy. A sural nerve biopsy performed early in the disease showed axonal degeneration with a virtual absence of unmyelinated fibres and moderate loss of myelinated fibres, mainly affecting the small fibres; this differs from previous reports. An immune-mediated or direct virus action might explain the pathogenesis of this unusual evolution of a viral infection.


Mouse peritoneal macrophage prostaglandin E1 synthesis is altered by dietary gamma-linolenic acid. The ability of dietary gamma-linolenic acid [18:3(n-6)] to modulate prostaglandin biosynthesis in mouse resident peritoneal macrophages was determined. Mice were fed diets containing corn oil, borage oil or evening primrose oil or a mixture of borage and fish oils. After 2 wk, resident peritoneal macrophages were isolated and stimulated with unopsonized zymosan to induce prostaglandin synthesis. Borage oil, primrose oil and fish-borage oil mixture dietary groups (containing 25.6, 11.9 and 19.5 g gamma-linolenic acid/100 g fatty acids, respectively) had significantly (P less than 0.05) enhanced prostaglandin E1 synthesis (39.7, 29.4 and 73.0 nmol prostaglandin E1/mg protein, respectively) compared with corn oil-fed (containing less than 0.1 g gamma-linolenic acid/100 g fatty acids) animals, which synthesized less than 0.1 nmol prostaglandin E1/mg protein. Borage oil- and fish-borage oil mixture-fed mice had the highest biosynthetic ratio of prostaglandin E1/prostaglandin E2 (E1/E2 approximately 0.2). Macrophages from borage oil-fed mice synthesized the lowest amount of prostacyclin (198.7 nmol 6-keto-prostaglandin F1 alpha/mg protein) compared with corn oil-, primrose oil- and fish-borage oil mixture-fed mice (379.7, 764.8 and 384.2 nmol 6-keto-prostaglandin F1 alpha/mg protein, respectively). In addition, borage oil-, primrose oil- and fish-borage oil mixture-fed mice had significantly (P less than 0.05) higher levels of dihomo-gamma-linolenic acid [20:3(n-6)] in membrane phospholipids (5.5, 3.5 and 5.7 mol/100 mol, respectively) relative to corn oil-fed mice (2.0 mol/100 mol).(ABSTRACT TRUNCATED AT 250 WORDS)


Follow-up evaluation of cervicovaginal human papillomavirus infection in adolescents. We performed a second examination for human papillomavirus (HPV) DNA on 51 sexually experienced 13- to 21-year-old (mean = 17.8 years) female patients seen at an urban teaching hospital. Cervicovaginal lavages were performed 6 to 36 months apart (median = 13.3 months) to collect cells for HPV DNA detection and typing by Southern blot hybridization. At the first and second visits, 39.2% (20/51) and 25.5% (13/51) of patients, respectively, were infected with HPV. Collectively, 56.9% (29/51) of patients had at least one positive HPV test result. Although 7.8% (4/51) were infected with HPV at both visits, only one patient had infection with the same HPV type. These findings suggest that although HPV infection is a common sexually transmitted disease, genotype-specific HPV infection detected by Southern blot at two visits was rare.


Demonstration of calcitonin gene-related peptide receptors in the gubernaculum by computerized densitometry. The gubernaculum appears to guide inguinoscrotal testicular descent by migration into the scrotum ahead of the testis. Calcitonin gene-related peptide (CGRP) has been found in the scrotal branches of the genitofemoral nerve of neonatal rats, and is known to stimulate gubernacular motility in vitro. This study aimed to identify CGRP receptors in the gubernaculum, which should be present if CGRP mediates gubernacular migration toward the scrotum. Gubernacular sections from neonatal male rats were incubated with [125I]-human CGRP as well as a variety of unlabeled neuropeptides. By using computerized densitometry, the quantitation of CGRP binding derived from in vitro autoradiography demonstrated a distinctive distribution of binding sites for CGRP over the developing cremasteric muscle in the gubernaculum. The binding analysis showed a single class of sites with a dissociation constant (Kd) of 2.13 nmol/L and a receptor density of 27.4 fmol/mg polymer. These results endorse the hypothesis that CGRP released from the nerve acts directly on the cremaster via its own receptors, which have not been described previously.


A strategy for resection of Wilms' tumor with vena cava or atrial extension. Resection of a Wilms' tumor that extends into the vena cava or right atrium results in excellent survival when combined with adjuvant therapy. Preoperative identification of the presence of intravascular tumor thrombus and the level of vascular involvement is essential. It facilitates safe surgical resection, with cardiopulmonary bypass immediately available for retrohepatic and atrial tumors. Six patients with intracaval or intracardiac tumor thrombus were treated over a 5-year period with no perioperative deaths. Preoperative chemotherapy was useful in two patients with extensive tumors and pulmonary metastases. Our results using an integrated management plan suggest that an aggressive surgical approach is justified for this extensive variant of Wilms' tumor.


Breastfeeding success with preterm quadruplets. Although the incidence of triplet and quadruplet birth has increased in the United States, few research-based guidelines are available for assisting mothers of these multiple births with breastfeeding. The purpose of this case study is to report a successful breastfeeding experience of a mother with preterm quadruplets. The quadruplets were born by cesarean delivery at 34 weeks' gestation and weighed from 1,820 g to 2,240 g. In-hospital breastfeeding experiences were managed by the authors, according to research-based guidelines for breastfeeding preterm neonates and infants. During the first month after discharge of the four newborns, the mother breastfed 12-34 times daily. Mean daily weight gains for the quadruplets during this time varied from 30 g to 54 g, indicative of adequate maternal milk supply. Nurses in maternity and neonatal specialties can apply the findings from this study to similar cases of mothers who want to breastfeed multiple neonates or infants.


[Magnetic resonance imaging of malignant hepatic tumors in adults]. Recently, magnetic resonance imaging (MRI) had an important place in imaging techniques of malignant primitive tumors of the liver. For many authors, this technique appears as the reference modality for hepatic tumor evaluation. Nevertheless, MRI is considered to be primarily a problem-solving rather than a screening modality. This specific goal of this technique is due to a more precise characterization of the lesions compared to other imaging modalities. The authors, according to recent data in literature and their own experience, present MRI findings of the more frequent malignant hepatic tumors encountered in adult.


[Role of MRI in the diagnosis of endocrine tumors of the pancreas]. Eight patients affected with endocrine tumor of the pancreas were examined, within the same period of time, by MRI and CT. Results from those two examinations were similar for the detection of the primary tumor (succeeding to visualize the lesion 5 times out of 8) and the evaluation of locoregional and vascular extension. No tumor smaller than 3 cm was diagnosed by MRI. Most of cases the pancreatic tumor appeared as hypointense in T1 and hyperintense in T2 sequences. MRI was a little more efficient than CT for the detection of liver metastases. MRI seems to be an interesting method for the follow-up of those patients needing a regular and prolonged surveillance after primary tumor ablation.


Juvenile nasopharyngeal angiofibroma: a trap for the unwary. Juvenile nasopharyngeal angiofibroma (JNA) is a rare, benign but aggressive tumour of the nasopharynx. It occurs primarily in male adolescents. The usual presentation is either nasal obstruction or epistaxis. We present a case with complete nasal obstruction of short duration. The management is discussed with reference to the literature. Because this tumour tends to occur in men in their second decade it is an important disease to consider in soldiers.


Protein interactions with urea and guanidinium chloride. A calorimetric study. The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.


Interferon-gamma-like immunoreactivity in sensory neurons may influence the replication of Sendai and mumps viruses. Rat dorsal root ganglia in tissue culture, which contain an interferon-gamma (IFN-gamma)-like immunoreactive subpopulation of neurons, were infected with paramyxoviruses. Sendai virus caused a substantial neuronal lysis, while the RW strain of mumps virus caused a much less pronounced nerve cell loss. Early during infection, the subpopulation of IFN-gamma-like immunoreactive neurons was less susceptible to mumps virus. Virus antigen was rapidly lost from surviving IFN-gamma-like positive neurons infected with Sendai virus, while this remarkable self-curing effect occurred in both nerve cell populations at later time points after mumps virus infection. By quantitative enzyme-linked immunosorbent assay (ELISA) technique, increased levels of "neuronal IFN-gamma" were recorded at 10 hr and 30 hr after infection with Sendai and mumps virus, respectively. This study indicates a role for the neuronal IFN-gamma-like molecule in determining the outcome of a viral infection in sensory ganglia.


Subcellular distribution of ubiquitin-protein conjugates in the hippocampus following transient ischemia. Ubiquitin-protein conjugates in the hippocampus were analyzed by immunoblotting with a monoclonal anti-ubiquitin antibody. In the CA1 region, Triton X-100 insoluble ubiquitin-protein conjugates increased after 24 hr following 20 min of ischemia. When the total hippocampi were fractionated subcellularly, ubiquitin-protein conjugates increased in the particulate, especially in the mitochondrial fraction. The ubiquitin-protein conjugates were solubilized by SDS, or were partially solubilized by urea. The results indicate that insoluble ubiquitin-protein conjugates increase after ischemia.


Differential coupling of opioid binding sites to guanosine triphosphate binding regulatory proteins in subcellular fractions of rat brain. In this study, we present evidence for the occurrence of mu, delta, and kappa opioid binding sites in synaptic plasma membranes (SPM) and microsomes of rat brain. Binding to all three opioid classes was inhibited by 5'-guanylylimidodiphosphate (Gpp[NH]p) in SPM, while microsomal sites proved to be insensitive to this GTP analog. Sensitivity was restored upon solubilization of microsomes with digitonin, suggesting that opioid receptors are physically separated from G proteins in this fraction. Modulation of microsomal binding by Na+ and Mn++ was greater than that of SPM. Pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation revealed the presence of G proteins with alpha-subunit molecular weights of 40 kDa in both subcellular fractions. Basal low Km GTPase activity in SPM was greater than in microsomes. Etorphine elicited a concentration-dependent stimulation of guanosine triphosphatase (GTPase) activity in SPMs but not in microsomes, indicating functional coupling of opioid receptors to G protein in the former and an uncoupling in the latter. Microsomes from 3-day-old rat brain contained more mu opioid sites and they were more sensitive to Gpp(NH)p inhibition than those in adults. These results are consistent with the hypothesis that opioid binding sites in adult microsomes are internalized and G protein uncoupled, while those in neonates are newly synthesized, coupled receptors.


Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. The use of autologous urothelium would be advantageous for urothelial replacement in many genitourinary reconstructive procedures. Urothelial tissue grafts might be created using isolated populations of transitional epithelium or tissue in concert with an appropriate synthetic substrate. We describe the results of experiments designed to determine the feasibility of using biodegradable polymers as delivery vehicles for the creation of new urothelial structures in vivo from dissociated cells. Primary cultures enriched in uroepithelial cells were obtained from New Zealand white rabbits using a new technique of cell harvest. Cells were seeded onto nonwoven meshes of polyglycolic acid polymers in culture and, after 1 to 4 days in vitro, the cell-polymer scaffolds were implanted into the mesentery, omentum or retroperitoneum of athymic mice. Polymers implanted without cells served as controls. Animals were sacrificed at 5, 10, 20 and 30 days after implantation and 75 implants were examined histologically. Ten days after implantation isolated single cell layers were seen lining the polymer fibers. At 20 and 30 days polymer degradation was evident and urothelial cells lined the polymer in continuous layers of 1 to 3-cell thickness. Anticytokeratin western blots demonstrated the presence of a urothelium-associated cytokeratin in cell-polymer implants recovered after 30 days. These results demonstrate that urothelial cells can be successfully harvested, survive in culture and attach to artificial biodegradable polymers. The urothelial-polymer scaffolds can be implanted into host animals and the implanted cells can achieve spatial orientation as the polymer undergoes biodegradation. These findings suggest that it may be possible to use autologous urothelium, reconfigured on a synthetic substrate, in reconstructive procedures involving the ureter, bladder and urethra.


Presence of lower temperature-dependent antibody with low avidity to C100-3 (HCV) antigen in voluntary blood donors. Serum samples with lower temperature-dependent antibody with low avidity to C100-3 (HCV) antigen were found in 0.19% of 23,197 voluntary blood donors at this blood center. They showed positive C100-3 antibody activity at 24 C but not at 37 C. The antibody activity bound to C100-3 antigen at lower temperature disappeared after incubation for 60 min at 37 C or treatment with 8 M urea. Other markers of hepatitis C virus infection, especially the presence of HCV-RNA were demonstrated in some of these serum samples and the importance of this phenomenon is discussed with regard to virus screening of blood donors for hepatitis C.


Ca(2+)-channel blockade in rat thoracic aorta by protopine isolated from Corydalis tubers. The pharmacological properties and mechanism of the action of protopine on isolated rat thoracic aorta were examined. It inhibited norepinephrine (NE, 3 microM)-induced tonic contraction in rat thoracic aorta in a concentration-dependent manner (25-100 micrograms/ml). The phasic contraction caused by NE was inhibited only by a high concentration of protopine (100 micrograms/ml). At the plateau of NE-induced tonic contraction, the addition of protopine also caused relaxation. This relaxing effect of protopine was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and it still existed in denuded rat aorta or in the presence of nifedipine (2-100 microM). Protopine also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03-3 mM) contraction of rat aorta in a concentration-dependent manner. Neither cAMP nor cGMP level was changed by protopine. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine were also not affected by protopine. 45Ca2+ influx caused by either NE or K+ was inhibited by protopine concentration-dependently. It is concluded that protopine relaxed the rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.


Effects of L-NG-monomethyl arginine on the cyclic GMP formations in rat mesenteric arteries. To evaluate the contribution of L-arginine as a precursor of the endothelium-derived relaxing factor (EDRF) on vascular cyclic GMP formation, we examined the effects of L-NG-monomethyl arginine (L-NMMA), and analog of L-arginine, on basal and acetylcholine (ACh)-, sodium nitroprusside (SNP)- and atrial natriuretic peptide (ANP)-induced cyclic GMP formations in rat mesenteric arteries. The mesenteric arteries were perfused with Krebs-Henseleit solution containing 0.2 mM isobutyl methyl xanthine. The effluents from the arteries were collected before and after infusions of graded doses of ACh, SNP or ANP in the absence or presence of 100 microM L-NMMA, and the levels of cyclic GMP were measured. Basal and ACh-induced cyclic GMP formations in the mesenteric arteries were significantly inhibited in the presence of L-NMMA, whereas a concomitant infusion of 300 microM L-arginine restored the inhibition of basal as well as ACh-induced cyclic GMP formations. L-NMMA did not affect SNP- and ANP-stimulated cyclic GMP formations, respectively. These results suggest that L-arginine is necessary for not only the stimulated cyclic GMP formation but also the basal cyclic GMP formation in the mesenteric arteries, whereas the SNP- and ANP-stimulated cyclic GMP formations in the arteries are independent of L-arginine.


Role of PGE2 in neurotransmission from pre- to post-ganglionic hypogastric nerves of guinea pigs. The hypogastric nerve to guinea pig vas deferens was stimulated pre- or post-ganglionically by adjusting the position of the suction electrode. Both stimulations induced a biphasic contraction consisting of a rapid transient phase and a delayed tonic phase. Indomethacin partially inhibited the contraction induced by pre-ganglionic stimulation, but did not inhibit that induced by post-ganglionic stimulation. Prostaglandin (PG) E2 counteracted the inhibitory effect of indomethacin. Mepacrine also inhibited the contraction induced by pre-ganglionic stimulation. Arachidonic acid and PGE2 both reversed the inhibition. The PGE2-receptor antagonist SC-19220 inhibited the contraction induced by pre-ganglionic, but not post-ganglionic nerve stimulation. These results suggested that endogenous PGE2 is important in neurotransmission in the pelvic ganglion of guinea pigs.


Influences of postnatal age and dietary nucleotides on plasma fatty acids in the weanling rat. Dietary nucleotides seem to play a number of physiologic roles during early life. They are improved in the maintenance of the immune system, intestinal maturation, and lipid metabolism. Nucleotides affect the conversion of essential fatty acids into their long-chain polyunsaturated (PUFA) derivatives in both preterm and at-term newborn infants. This work examines the effect of postnatal age and dietary nucleotides on the fatty acid composition of total plasma lipids and lipid fractions in the rat. Weanling rats (21 days old) were divided into three groups. The first group was killed, and the other two groups were fed a standard semipurified diet, and the same diet supplemented with 250 mg each of CMP, UMP, AMP, GMP, and IMP per 100 g of diet for 4 weeks. Advancing postnatal age led to an increase of total plasma fatty acids, especially saturated, and PUFA of the n-6 series, whereas PUFA of the n-3 series decreased. The fatty acid profile of plasma phospholipids (PL) exhibited minor changes, although there was a tendency to show lower levels of saturates and PUFA of the n-3 series and increased levels of PUFA of the n-6 series. Cholesteryl esters showed a response similar to that of PL, although the increase in arachidonic acid (20:4n-6) was significant. For triglycerides, linoleic acid (18:2n-6) and monounsaturates increased their levels, whereas saturates decreased. Dietary nucleotides mediated a significant increase in total plasma fatty acids, namely monounsaturated fatty acids and PUFA of both n-6 and n-3 series as compared with the control group. The relative fatty acid composition of PL and cholesteryl esters was mostly unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibitory effect of NMDA on dopaminergic transmission in a slice preparation of rat globus pallidus. The effect of N-methyl-D-aspartate (NMDA) on KCl-evoked endogenous dopamine (DA) release from slices of rat globus pallidus (GP) was examined. NMDA inhibited the KCl-evoked DA release in a dose-dependent manner. This inhibition was blocked by CPP, an NMDA antagonist. Tetrodotoxin partially antagonized the effect of NMDA. The NMDA-induced inhibition was also partially antagonized by bicuculline methiodide and was mimicked by muscimol. These results strongly suggest that 1) an activation of NMDA receptors exerts an inhibitory effect on dopaminergic transmission in GP and 2) GABAergic transmission is involved in the effect of NMDA.


The use of the saddle prosthesis for reconstruction of the hip joint after tumor resection of the pelvis. Reconstruction of the hip joint by a saddle prosthesis after excision of a malignant pelvic tumor is a relatively new method, which thus far has been mainly used for revision of infected hip arthroplasties. One patient with a metastatic cystosarcoma phyllodes and one patient with a chondrosarcoma of the pelvis were treated by local resection and reconstruction with a saddle prosthesis. Although the patient with the metastatic cystosarcoma phyllodes died 9 months after surgery due to metastatic disease, both patients had early recovery, with no difference in leg length and obtained early painless complete weight bearing with satisfactory functional result. These two case reports clearly illustrate the usefulness of the saddle prostheses in limb saving surgery for malignant tumors of the pelvis.


Activity of sparfloxacin against Mycobacterium leprae inoculated into footpads of nude mice. The antileprosy activity of a new quinolone, sparfloxacin, was examined in the nude mouse footpad model. By serial dosing (once a day, 5 or 6 times per week, during the 3rd-5th months postinoculation), 10 mg/kg of sparfloxacin displayed bactericidal-type activity and bacteriostatic activity was present at daily doses of 5 and 2 mg/kg. By intermittent dosing (once a day, twice weekly at daily doses of 10 and 20 mg/kg or once weekly at a daily dose of 30 mg/kg, during the 3rd-5th months postinoculation), sparfloxacin markedly inhibited the growth of leprosy bacilli with slight remultiplication at later stages. Sparfloxacin seems to be worth studying clinically as a novel antileprosy drug.


Fentanyl receptor assay. II. Utilization of a radioreceptor assay for the analysis of fentanyl analogs in urine. A radioreceptor assay has been developed to measure fentanyl and fentanyl-like drugs in biological specimens. The assay is based on the competition of these drugs with [3H]fentanyl for opioid receptors. Rats were injected intravenously with fentanyl (15 micrograms/kg), alpha-methylfentanyl (15 micrograms/kg), (+/-)-cis-3-methylfentanyl (15 micrograms/kg), butyrylfentanyl (0.48 mg/kg), and benzylfentanyl (3.19 mg/kg). Urine samples were analyzed by radioreceptor assay (RRA), radioimmunoassay (RIA), and gas chromatography/mass spectrometry (GC/MS). The time-course of urinary analysis of fentanyl analogs showed some discrepancies. RRA measurement of urine concentrations of (+/-)-cis-3-methylfentanyl that were undetectable by RIA gave results 5-10 times higher than values obtained by GC/MS. Concentrations of alpha-methylfentanyl obtained by RRA and GC/MS were similar; however, these samples were negative by RIA. Following the administration of benzylfentanyl, urinary concentrations were not detected by RIA and only slightly detectable with RRA; however, high concentrations of benzylfentanyl were found by GC/MS in the same samples. Urine samples from animals injected with butyrylfentanyl showed high cross-reactivity with fentanyl antibody, giving values measured by radioimmunoassay about two times higher than those obtained by the other two methods. These findings suggest that this radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine and correlates well with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs.


2-Bromomelatonin: synthesis and characterization of a potent melatonin agonist. A practical synthesis of N-[2-(2-bromo-5-methoxy-1H-indol-3-yl)ethyl]- acetamide (2-bromomelatonin) was achieved by direct bromination of melatonin with N-bromosuccinimide (NBS) in anhydrous acetic acid at room temperature under nitrogen, followed by flash-chromatography. 1H-NMR and mass spectra showed the bromine to be incorporated at the C-2 position of the indole moiety. Tests performed in vitro with isolated melatonin receptors from rabbit parietal cortex demonstrated that the relative binding affinity of 2-bromomelatonin was about ten times higher than that of melatonin and close to that of 2-iodomelatonin. 2-Bromomelatonin behaved as a potent agonist in the physiological studies. It showed enhanced activity in inhibiting the spontaneous firing activity of cortical neurons and similarly to melatonin and 2-iodomelatonin potentiated significantly the inhibitory effect of GABA. 2-Bromomelatonin was also an extremely effective agonist in the tests performed in vivo in the Syrian hamster gonadal regression model.


V1-receptor mediated GSH efflux by vasopressin from rat hepatocytes. Vasopression increases sinusoidal efflux of GSH in the perfused rat liver. The mechanism of this effect was studied in the perfused rat liver and in isolated rat hepatocytes. Vasopressin stimulated GSH efflux in both systems and a V1-receptor antagonist (OPC-21268) significantly inhibited the effect of vasopressin suggesting that vasopressin stimulates GSH efflux from rat hepatocytes via V1-receptor.


Assessment of dermal exposure of greenhouse workers to the pesticide bupirimate. An HPLC method was developed for estimation of dermal exposure of greenhouse workers to the pesticide bupirimate. Chromatography was performed on a cyano-modified silica column with methanol-water (6:4 by volume) containing 5 g/L ammonium sulfate as eluent. UV detection at 310 nm was used for quantitation. Dermal exposure was assessed by letting the workers wear cotton gloves and by measuring foliar dislodgable residues in the greenhouses as potential exposure. The analytical procedure was validated for measurement of bupirimate on cotton gloves and in solutions used for the estimation of foliar dislodgable residues. Gloves were extracted with methanol. Recovery of bupirimate from fortified gloves was complete. Methanol extracts with one volume of water added and solutions containing dislodgable residues were injected directly onto the HPLC system. The limit of detection was 30 micrograms/L. Between-day coefficients of variation were 7 and 4% at concentrations of 0.6 and 28 mg/L, respectively.


Excitatory amino acid signal transduction in the hippocampus: role of noradrenergic afferents and nitric oxide in cGMP increases in vivo. Previous studies have demonstrated that excitatory amino acid (EAA)-dependent increases in cerebellar cGMP are dependent upon the prior activation of nitric oxide (NO) synthase. Additionally, the actions of NMDA, but not kainate or quisqualate, in elevating cerebellar cGMP have been shown to be dependent upon intact noradrenergic innervation of the cerebellum. In the current study we extended these observations to the hippocampus and again found that EAA-dependent increases in hippocampal cGMP also involve prior formation of NO. And as in the case of the cerebellum, NMDA-dependent increases in hippocampal cGMP involve prior release of norepinephrine which in turn apparently activates an alpha 1-adrenergic receptor to elicit cGMP increases. In toto, these data suggest that a key role of NMDA receptors in these brain regions is to presynaptically regulate the release of norepinephrine, thereby modulating the tone of this monoaminergic system. This may be a general principle which needs experimentation in other terminal fields of noradrenergic pathways.


A system of transposon mutagenesis for bacteriophage T4. We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.


Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid. The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.


Effects of mental stress on lipolysis in humans. Lipid mobilization was investigated in subcutaneous adipose tissue specimens obtained before and after a standardized mental-stress test in 14 non-obese healthy subjects. All participants responded with an increased heart rate and elevation of plasma glycerol levels. Plasma norepinephrine concentrations remained unchanged throughout the test. In six subjects, mental stress induced a significant increase in plasma epinephrine levels, to more than 0.26 nmol/L (responders), while the remaining eight individuals showed a response of less than 0.12 nmol/L (nonresponders). In the responders, a 30% increase in catecholamine-stimulated in vitro lipolysis was found after the mental-stress test, while the lipolytic response in isolated fat cells in vitro decreased slightly in the nonresponders after mental stress. A strong correlation (r = .84) was observed between the increased in vitro lipolytic responsiveness due to mental stress and circulating plasma epinephrine levels. In vitro data suggest that the augmentation in lipolytic activity induced by acute mental stress was caused by alterations between the beta-adrenoceptor and adenylate cyclase, ie, probably an increased coupling between beta-receptors and the stimulatory guanosine triphosphate [GTP]-binding protein (G2). This, in combination with elevated levels of circulating epinephrine, may explain the increased lipolysis during mental stress in some individuals (ie, responders). However, other parallel mechanisms for activation of lipolysis during mental stress must also exist in certain individuals (ie, nonresponders), and seem not to involve the adrenergic system.


Meeting the challenge of multidrug-resistant tuberculosis: summary of a conference. On January 22-23, 1992, a conference on "Meeting the Challenge of Multidrug-Resistant Tuberculosis" (MDR-TB) was held at the Centers for Disease Control, with greater than 400 attendees. Plenary presentations summarized the nature and magnitude of the problem, and work groups were formed to address six issues: public health, epidemiology and surveillance, infection control and environment, laboratory diagnosis and research, therapy, and training and education. Participants concluded that existing tools for diagnosis, patient management, and infection control must be more thoroughly implemented and that research to develop new approaches to diagnosis and therapy is critically needed. Discussions at the conference provided information that has been used to develop the National Action Plan to Combat Multidrug-Resistant Tuberculosis.


Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport. Effects of endotoxin administration on the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca2+ transport in cardiac sarcolemma was decreased by 22 to 46% (p less than 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the Vmax for ATP and for Ca2+ were decreased by 50% (p less than 0.01) and 32% (p less than 0.01), respectively, while the Km values for ATP and Ca2+ were not significantly affected after endotoxin administration. Magnesium (1-5 mM) stimulated while vanadate (0.25-3.0 microM) inhibited the ATP-dependent Ca2+ transport, but the Mg(2+)-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca2+. Additional experiments show that the Ca2+ sensitivity of the Ca(2+)-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca2+ pumping without affecting Ca(2+)-ATPase activity. Since sarcolemmal ATP-dependent Ca2+ transport plays an important role in the regulation of cytosolic Ca2+ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca2+ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.


Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport. The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca2+ transport in canine cardiac sarcolemma was decreased by 30-35% 4 h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca2+ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca2+ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A2 inhibited Ca2+ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca2+ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.


Regulation of calcium transport in pancreatic acinar plasma membranes from guinea pig. The regulation of the guinea-pig pancreatic acinar plasma membrane Ca2+ pump by protein kinase A, protein kinase C and calmodulin was investigated. The results were compared with the effects of these regulators on the high affinity Ca(2+)-ATPase found in this membrane preparation. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca2+ transport 2-fold, but had no effect on Ca(2+)-dependent ATPase activity. Purified protein kinase C, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate and diacylglycerol derivative, 1-stearoyl-2-arachidonoyl-sn-glycerol, failed to stimulate the Ca(2+)-uptake but augmented the Ca(2+)-dependent ATPase activity. Exogenously added calmodulin failed to stimulate either activity. In addition, two antagonists of calmodulin activity, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca(2+)-transport. These data suggest the presence of endogenous calmodulin within guinea-pig pancreatic acinar plasma membranes. Both calmodulin antagonists failed to influence the Ca(2+)-dependent ATPase activity. The ability of boiled extracts from guinea-pig pancreatic acinar plasma membranes to stimulate the Ca(2+)-ATPase activity in calmodulin-depleted erythrocyte plasma membranes confirmed the presence of endogenous calmodulin. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca2+ efflux from pancreatic acinar cells. These results also provide further evidence suggesting that the high affinity Ca(2+)-ATPase does not catalyze the plasma membrane Ca(2+)-transport activity observed in pancreatic acini.


Oxygen free radical producing activity of polymorphonuclear leukocytes in patients with Parkinson's disease. Oxygen free radicals (OFRs) have been suggested in the pathogenesis of Parkinson's disease (PD). These free radicals exert their cytotoxic effect by peroxidation of lipid membrane resulting in the formation of malondialdehyde (MDA). Polymorphonuclear (PMN) leukocyte is one of the major sources of OFR. However, the oxygen free radical producing activity of PMN leukocytes in patients with PD is not known. We therefore studied the oxygen free radical producing activity of polymorphonuclear leukocytes and MDA levels in the serum of healthy subjects and in patients with Parkinson's disease. The oxygen free radical producing activity of PMN leukocytes in blood and the MDA content in serum were significantly higher in patients with Parkinson's disease than in healthy subjects. These results indicate a possible role of oxygen free radicals in the pathogenesis of Parkinson's disease.


Homologous desensitization of calcitonin gene-related peptide response in rat glomerular mesangial cells in culture. Addition of calcitonin gene-related peptide (CGRP) to rat glomerular mesangial cells in culture resulted in an increase in cyclic adenosine monophosphate (cAMP) accumulation in a concentration-dependent fashion with an EC50 of approximately 3 nM. Inclusion of CGRP8-37, a CGRP1 selective antagonist shifted CGRP concentration-response curve to the right, suggesting the presence of CGRP1 subtype receptors in these cells. Pretreatment of these cells with CGRP followed by washing and rechallenge with CGRP or isoproterenol or forskolin resulted in selective attenuation of CGRP-mediated cAMP accumulation by 55-60% without affecting isoproterenol or forskolin-mediated accumulation, suggesting that CGRP pretreatment of mesangial cells induced homologous desensitization. CGRP-mediated desensitization of cAMP accumulation was found to be both concentration- and time-dependent. Desensitization did not affect the EC50 of CGRP for stimulation of cAMP accumulation, but resulted in a 55-60% reduction in maximal response. CGRP-mediated desensitization was fast, with half-maximal desensitization occurring as early as 5 min after pretreatment with CGRP. In addition, CGRP-mediated desensitization was blocked by the CGRP receptor antagonist, CGRP8-37, when included in the pretreatment protocol. These data suggest that rat mesangial cells display CGRP1 subtype receptors and that prolonged pretreatment of these cells with CGRP resulted in homologous desensitization.


Glycosphingolipids: the putative receptor for Staphylococcus aureus enterotoxin-B in human kidney proximal tubular cells. We have investigated the binding of 125I-staphylococcal enterotoxin-B (SEB) in cultured human proximal tubular cells. We found that the binding of 125I-SEB to PT cells was time and concentration dependent and competitively inhibited by antibody against SEB. Preincubation of cells with trypsin and neuraminidase or with fetuin did not significantly impair the binding of 125I-SEB to such cells. In contrast, treatment with endoglycoceramidase completely inhibited the binding of 125I-SEB to cells. Neutral glycosphingolipids exerted a concentration-dependent inhibition of 125I-SEB binding to such cells, maximum inhibition (96% compared to control) occurred upon incubation of PT cells with neutral glycosphingolipids. Taken together, our studies indicate that SEB specifically binds to a neutral glycosphingolipid in PT cells. In contrast, staphylococcal enterotoxin-A and toxic shock toxin (TST-1) are bound to a protein in such cells.


Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase. We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99-126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition (approximately 45%) was observed in the presence of 10-30 microM GTP gamma S, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na+ enhanced the degree of inhibition by about 60%, whereas K+ and Li+ suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn2+ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 mM, and at 2 mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A2 treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca(2+)-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.


Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Introduction of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis. Ca2+ however regulates functions not only in the cytosol but also within various organelles where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photoprotein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.


Association of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha beta T-cell receptor. The membrane glycoprotein CD4 enhances antigen-mediated activation of T cells restricted by class II molecules of the major histocompatibility complex (MHC). This positive function has been attributed to the protein tyrosine kinase p56lck (ref. 4), which is noncovalently associated with the cytoplasmic portion of CD4, and is activated on CD4 aggregation. Antigen presentation by MHC class II molecules coaggregates CD4 and the T-cell antigen receptor (TCR alpha beta-CD3). Thus, the mutual specificity of CD4 and TCR alpha beta for the MHC-antigen complex results in the juxtaposition of p56lck and TCR alpha beta-CD3. In contrast, anti-CD4 antibodies can abrogate antigen-induced, as well as anti-TCR-induced T-cell activation, indicating that CD4 might also transduce negative signals. The molecular basis for this opposing function remains unclear. Here we show that the CD4-p56lck complex prohibits the induction of activation signals through the TCR-CD3 complex when not specifically included in the signalling process. This negative effect does not require anti-CD4 treatment, indicating that the induction of distinct negative signals is probably not involved. Rather, the results demonstrate that the CD4-p56lck complex provides prerequisite signals for antigen-receptor-induced T-cell growth and thus characterize a molecular mechanism for functional constraints imposed on T-cell activation by the MHC.


Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Transcription of the proto-oncogene c-fos is stimulated rapidly and transiently by serum growth factors and mitogens. Critical for this response is the serum-response element which is bound in vivo in a ternary complex containing the transcription factors p67SRF and p62TCF (ref. 2). Disruption of the ternary complex correlates with impaired induction by serum and phorbol ester. Mitogen-activated protein (MAP) kinase is a serine/threonine kinase which is activated 1-5 minutes after treatment of cells with mitogens and growth factors that induce re-entry into the cell cycle, making MAP kinase a candidate for the transmission of proliferative signals. Here we show that p62TCF is phosphorylated by MAP kinase in vitro and that phosphorylation results in enhanced ternary complex formation. Serum-starved Swiss 3T3 cells treated with epidermal growth factor, which induces MAP kinase in these cells, are induced to express c-fos and yield p62TCF active in ternary complex formation. In contrast, treatment of Swiss 3T3 cells with insulin, which does not activate MAP kinase under these conditions, does not lead to enhanced ternary complex formation nor does it induce c-fos transcription. Our results link the expression of the human c-fos proto-oncogene to signal transduction pathways known to be activated before its own induction.


Raf-1 activates MAP kinase-kinase.

Raf-1 activates MAP kinase-kinase. The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.


Different beta-subunits determine G-protein interaction with transmembrane receptors. Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.


[A surprising double tumor of the liver 60 years following thoratrast diagnosis]. A 66-year-old man with thorotrastosis of the reticuloendothelial system is described. Post mortem two tumours were found in an enlarged liver: a cholangiocarcinoma and an angiosarcoma. Coincidentally, a hilar neurofibroma was found. The former two tumours most probably developed because of a lifelong endogenous alpha-irradiation by thorium disintegration. The exceptional latency period of 60 years' duration is emphasized.


Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism. Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats, confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.


Effects of morphine and D-Ala2-D-Leu5-enkephalin in the seizure-susceptible El mouse. Opioid agonists were used to investigate the modulation of seizures in the seizure-susceptible El mouse. Morphine and D-Ala2-D-Leu5-enkephalin (DADLE) were injected subcutaneously or intracisternally as prototypic agonists for mu and delta opioid receptors. Systemic or intracisternal injection of both morphine and DADLE decreased the incidence of seizures and the seizure score in El mice in a dose-dependent manner. The anticonvulsant effects of morphine and DADLE were reversed by naloxone (2 mg/kg, s.c.). This implies that opioid agonists have anticonvulsant properties which are mediated by mu and delta opioid receptors. In conclusion, a deficit in endogenous opioid peptides, which act as anticonvulsants may play a significant role in the etiology or pathophysiology of seizures in the El mouse.


Evidence that hypothalamic periventricular dopamine neurons innervate the intermediate lobe of the rat pituitary. The purpose of the present study was to provide neurochemical and endocrinological evidence that dopamine (DA) neurons terminating in the intermediate lobe of the rat pituitary originate in the periventricular nucleus of the hypothalamus. One week following surgical separation of the periventricular nucleus from the mediobasal hypothalamus, DA and 3,4-dihydroxyphenyl-acetic acid (DOPAC) concentrations in the intermediate lobe were reduced by 50%, and this was accompanied by an increase in plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentrations. In contrast, this procedure had no effect on concentrations of prolactin in the plasma, or DA or DOPAC in the median eminence, the region of the mediobasal hypothalamus containing terminals of tuberoinfundibular DA neurons. Electrical stimulation of the periventricular nucleus increased the ratio of DOPAC/DA in the intermediate lobe and reduced the concentrations of alpha-MSH in the plasma, whereas in these same animals the DOPAC/DA ratio in the median eminence and concentrations of prolactin in the plasma were unaltered. These results indicate that approximately 50% of all the DA neurons terminating in the intermediate lobe of the rat pituitary originate in or project through the periventricular nucleus of the hypothalamus, and that these DA neurons regulate the secretion of alpha-MSH from intermediate lobe melanotrophs.


Hormonal regulation of medial preoptic mu-opiate receptor density before and after parturition. Endogenous opioid peptides acting in the medial preoptic area (MPOA) appear to be involved in the regulation of maternal behavior in lactating rats. Moreover, it is known that the density of mu-opiate receptors in the MPOA is elevated during pregnancy, but decreases during lactation. In the first experiment of this study, mu-receptor density in the preoptic area was examined across the periparturitional period on gestation days 18, 20 and 22, at 1 h postpartum and on postpartum days 1 and 12. The effect of pregnancy during lactation on mu-receptor density was also assessed. In addition, plasma hormone concentration of estradiol (E2), progesterone (P), and prolactin (PRL) were determined. While plasma P levels decreased and PRL levels increased prior to parturition, MPOA mu-receptor density remained elevated until 24 h after parturition before declining to reach a level similar to that of ovariectomized control animals. Receptor density was significantly correlated with PRL levels only in gestation day 22 animals, when PRL levels were highest. MPOA mu-receptor density was low at postpartum day 12 whether or not the animal was pregnant. No effects were observed in the adjacent lateral preoptic area in any group. In the second experiment, the effect of hormonal manipulation on preoptic opiate receptor density was examined at various times after removal of Silastic capsules containing P following sustained E2/P exposure. While P levels decreased abruptly following capsule removal, MPOA receptor density declined more gradually. The results are consistent with the hypothesis that a reduction of mu-receptor density occurs in the MPOA following parturition by receptor turnover in the absence of sufficient hormonal stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Hereditary neuropathy with liability to pressure palsies in childhood. Four children, index cases of families in which autosomal dominant neuropathy with liability to pressure palsies (HNPP) was diagnosed, are presented. Only one child was admitted for evaluation of an acute peroneal palsy, three presented with other symptoms. Polyneuropathy was diagnosed in all four children, in one of their parents and in some sibs. On inquiry, one child and several members of the four families had experienced transient palsies before. Morphological studies of the sural nerves showed frequently large tomacula and a neuropathic process of segmental de- and remyelination, and axonal degeneration. Attention is drawn to the atypical presentation without pressure palsies of HNPP.


Mitochondrial angiopathy in a family with MELAS. A family with mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike epidoses (MELAS) affecting mother, son and daughter is described. Biochemical studies on muscle biopsy specimen in one patient revealed NADH dehydrogenase (complex I) deficiency. A mitochondrial angiopathy could be demonstrated by brain and muscle biopsy. It is suggested that the mitochondrial angiopathy is the basic pathogenic mechanism of impaired cerebral circulation in MELAS.


Vasoactive intestinal polypeptide modulates neuronal excitability in hippocampal slices of the rat. Vasoactive intestinal polypeptide added at submicromolar concentrations to the perfusion fluid of rat hippocampal slices and slice cultures enhanced the excitability of CA1 and CA3 pyramidal cells in several ways. Specifically, cells were depolarized and the Ca(2+)- and cyclic AMP-dependent potassium conductance was blocked as demonstrated by reduction of the long-lasting afterhyperpolarization and the accommodation of firing. This was also found in tetrodotoxin-containing medium. In low Ca(2+)-high Mg2+ medium (in synaptic isolation) the firing rate was increased. Synaptic transmission was potentiated: extracellularly registered excitatory postsynaptic potentials and population spikes in response to stratum radiatum stimulation and intracellularly recorded excitatory postsynaptic potential-inhibitory postsynaptic potential sequences were enhanced. These results are in keeping with the known stimulation of adenylate cyclase by vasoactive intestinal polypeptide.


The role of dihydropyridine-sensitive Ca2+ channels in stimulus-evoked catecholamine release from chemoreceptor cells of the carotid body. The present study utilized an in vitro preparation of the rabbit carotid body, with tissue catecholamine stores labeled by incubation with 3H-tyrosine. The goal was to characterize pharmacologically the voltage-dependent Ca2+ channels present in the type I (glomus) cells of this arterial chemoreceptor organ, and to elucidate their role as pathways for Ca2+ entry. We found that release of 3H-dopamine induced by high external potassium was over 95% dependent on external calcium concentration and that this release was 90-100% inhibited by the dihydropyridine antagonists, nisoldipine and nitrendipine, and was potentiated by the dihydropyridine agonist, BayK 8644. Therefore, any stimulus-induced, calcium-dependent release of 3H-dopamine that was inhibited by nisoldipine and potentiated by BayK 8644, was considered to be supported by Ca2+ entry into the cells via voltage-dependent Ca2+ channels. Significant differences were observed in the release of 3H-dopamine induced by 75 vs 25 mM K+. On prolonged stimulation, release induced by 75 mM K+ was large and transient, whilst that induced by 25 mM K+, although more moderate, was sustained. The release elicited by 75 mM K+ was inhibited approximately 90% by 1.5 mM Co2+ or 625 nM nisoldipine, while release by 25 mM K+ was completely blocked by 0.6 mM Co2+ or 125 nM nisoldipine. Low PO2-induced release of 3H-dopamine was 95% dependent on Ca2+, and was inhibited by nisoldipine (625 nM) in a manner inversely proportional to the intensity of hypoxic stimulation, i.e. 79% inhibition at a PO2 of 49 Torr, and 20% inhibition at PO2 of 0 Torr. BayK 8644 potentiated the release induced by moderate hypoxic stimuli. Release elicited by high PCO2/low pH, or by Na(+)-propionate or dinitrophenol-containing solutions, was approximately 80% Ca(2+)-dependent, and the dihydropyridines failed to modify this release. It is concluded that type I cells possess voltage-dependent Ca2+ channels sensitive to the dihydropyridines, which in agreement with previous electrophysiological data should be defined as L-type Ca2+ channels. Calcium entry which supports the release of 3H-dopamine elicited by moderate hypoxia should occur mainly through these channels while the release induced by strong hypoxic stimuli will be served by Ca2+ entry which occurs in part via voltage-dependent Ca2+ channels, and in part through an additional pathway, probably a Na+/Ca2+ exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)


Mechanism through which GABAA receptor modulates catecholamine secretion from bovine chromaffin cells. The actions and mechanism of GABAergic modulation of catecholamine secretion from isolated bovine chromaffin cells were investigated. The GABAA receptor agonist muscimol induced a fast rise in cytosolic [Ca2+]. The mean peak increase was 290 +/- 30 nM over basal levels. The increase in cytosolic [Ca2+] was abolished in the absence of extracellular [Ca2+] and was blocked by the GABAA antagonist bicuculline and the dihydropiridine nifedipine. Muscimol also elicited the release of catecholamines and increased the bisoxonol fluorescence indicating a cell depolarization. The [Ca2+] entry was well correlated with muscimol-evoked catecholamine secretion. When cells were treated with muscimol and a second secretagogue, a biphasic behavior was revealed. Muscimol enhanced the catecholamine release evoked by low concentrations of nicotine or K+, whereas release obtained at high concentrations of nicotine or K+ was actually inhibited. When the muscimol effect on membrane potential was studied in the presence of low K+ or nicotine concentrations, an enhancement of the bisoxonol fluorescence was observed. This effect was reversed at high concentrations of both K+ and nicotine. Measurement of 36Cl- fluxes showed an increase in membrane permeability to Cl- during muscimol stimulation. The influx or efflux in Cl- was dependent on membrane potential. In normal conditions, with a K+ concentration of 5.4 mM, a Cl- efflux was observed by both radiometric techniques, with 36Cl- and by the use of the chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulphopropil)quinolinium, as indicator of intracellular Cl-. At high nicotine (20 mM) or K+ concentrations (105 mM) a Cl- influx was observed using 6-methoxy-N-(3-sulphopropil)quinolinium.(ABSTRACT TRUNCATED AT 250 WORDS)


Antimigraine drug sumatriptan increases blood flow velocity in large cerebral arteries during migraine attacks. Sumatriptan, a novel selective 5-hydroxytryptamine1d (5-HT1d) receptor agonist, which is highly effective in the acute treatment of migraine attacks, blocks dural neurogenic plasma extravasation and constricts cranial blood vessels in animal experiments. We measured intra- and extracranial blood flow velocities (BFV) with a transcranial Doppler device in 67 patients during a spontaneous migraine attack, before and after treatment with 3 mg or 6 mg subcutaneous sumatriptan or placebo. Sumatriptan, but not placebo, significantly increased BFV (cm/sec) in the internal carotid and middle cerebral arteries on both sides, without detectably changing the BFV in the common and external carotid arteries. The rise in BFV increased with the dose of sumatriptan, parallel to an increase in proportion of patients improved. There were no significant changes in heart rate, blood pressure, or respiratory frequency after treatment with sumatriptan. The increase in BFV probably reflects vasoconstriction of the large basal intracranial arteries, which may be a mechanism for the antimigraine action of sumatriptan.


Expression of monocyte activation antigen Mo3 on the surface of peripheral blood monocytes from patients with multiple sclerosis. Exposure of human peripheral blood monocytes (PBM) to phorbol esters, bacterial products, and cyclic adenosine monophosphate agonists is known to stimulate expression of a plasma membrane antigen ([Ag]; Mo3). Mo3 is recognized by two monoclonal antibodies, Mo3e (IgM), and Mo3f (IgG). Surface Mo3 is barely detectable by indirect immunofluorescence flow cytometry in nonstimulated monocytes. Mo3-positive monocytes have been found in inflammatory tissues, but increased surface expression of Mo3 in PBM has not been seen in any patient group. We report that PBM from patients with chronic progressive MS (CPMS) express increased Mo3. PBM from patients with other neurologic diseases and healthy controls express little measurable Mo3. No difference was seen in class II major histocompatibility complex Ag expression and in Mo2 (CD14) expression. Exposure of PBM to lipopolysaccharide (10 mg/ml) enhanced Mo3 expression in both MS patients and controls. Mo3 expression on CPMS PBM was not dependent on culture conditions. Taken together, our observations suggest that monocytes from patients with MS are stimulated in vivo to express activation Ag Mo3, but that Mo3-positive monocytes need not be upregulated for HLA-DR.


Melatonin potentiates cyclic AMP production stimulated by vasoactive intestinal peptide in human lymphocytes. The present paper demonstrates the effect of melatonin on cyclic AMP production in human lymphocytes from peripheral blood. Melatonin by itself did not influence cyclic AMP accumulation in these cells at any dose studied; however, the drug potentiated the effect of vasoactive intestinal peptide (VIP) on the cyclic nucleotide production. In the presence of physiological concentrations of VIP (either 1, 10 or 100 pM), melatonin potentiated cyclic AMP production. However, at high doses of VIP (either 1, 10 or 100 nM), melatonin exhibited no such effect. The results suggest that human lymphocytes are a target for melatonin and that it may participate, jointly with VIP, in the regulation of immune function.


Release of calcitonin gene-related peptide like-immunoreactivity induced by electrical field stimulation from rat spinal afferents is mediated by conotoxin-sensitive calcium channels. Electrical field stimulation (EFS, 10 V, 50 mA/cm2, 5-50 Hz, 1 ms pulse duration, 10 s train every 20 s for 5 min) produced a rapid and reproducible outflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) from superfused slices from the dorsal half of the rat spinal cord which is abolished by tetrodotoxin (TTX, 0.3 microM), in vitro capsaicin desensitization (10 microM for 30 min) and in Ca-free medium. The response was unaffected by ruthenium red (10 microM), indomethacin (10 microM) and nifedipine (1 microM) while it was abolished by omega-conotoxin (omega-CTX, 0.1 microM) and, in a naloxone-sensitive manner, by morphine (3 microM). Since CGRP release from capsaicin-sensitive afferents evoked by EFS in rat peripheral tissues is conotoxin-resistant, these findings provide direct evidence for a qualitative difference between central and peripheral endings of capsaicin-sensitive primary afferents in the mechanisms regulating transmitter release in the same species.


Up-regulation of [3H]DAMGO and [3H]DTLET opioid binding sites in laminae I-II of the spinal cord in intact and deafferented morphine-tolerant rats. Using quantitative autoradiography and selective opioid ligands, we have measured the effects of morphine-induced tolerance on [3H]DAMGO and [3H]DTLET binding sites in the superficial spinal dorsal horn (laminae I-II) of intact and deafferented rats (unilateral C4-T2 dorsal rhizotomy). In intact rats, the treatment induced an up-regulation of 26% and 39% for [3H]DAMGO and [3H]DTLET binding sites, respectively, without modification of receptor affinity. In deafferented rats, the treatment similarly induced an up-regulation of 31% and 29% for [3H]DAMGO and [3H]DTLET binding sites, respectively, on the contralateral side, and of 21% and 25%, respectively, on the ipsilateral side. These data demonstrate that the up-regulation induced by morphine tolerance is of similar magnitude for both presynaptic (on primary afferent fibers) and postsynaptic (on spinal neurons) opioid binding sites in the rat dorsal horn.


Dual effects of endothelin-1 on neurite outgrowth induced by 12-O-tetradecanoylphorbol-13-acetate. The vasoactive peptide endothelin-1 (ET-1) was found to bind to a single class of binding sites in chick embryonic sensory ganglia with a Kd of 67 +/- 5 pM. Treatment of ganglia explants with 100 pM ET-1 did not affect neuronal development, but when added together with 12-O-tetradecanoylphorbol-13-acetate (TPA) a synergistic stimulation of neurite outgrowth was observed. In contrast, 10 nM ET-1 inhibited TPA-induced neurite outgrowth. Both the stimulatory and inhibitory effects were not blocked by nifedipine, a Ca2+ channel blocker. These results suggest that ET-1 can modulate the process of neurite outgrowth and its effects are not dependent on voltage-gated Ca2+ channels.


GABAA sodium independent receptor sites in a strain of rats presenting generalized non-convulsive seizures. The role of gamma-aminobutyric acid (GABA), a major inhibitor neurotransmitter in the central nervous system (CNS), is well established in the genesis and the control of epilepsies. The purpose of this work was to study the binding parameters of the Na(+)-independent GABA receptors in the brain of a strain of rats presenting spontaneous generalized non-convulsive seizures. The high- and low-affinity binding sites were evaluated in cerebral cortex, cerebellum, and hippocampus using [3H]muscimol. No significant modification was observed for the Bmax and the Kd of high-affinity binding sites, although a slight decrease of Bmax was noted in the three brain areas in rats with seizures. Concerning the low-affinity binding sites, significant decreases were observed in the values of Bmax in the cortex, cerebellum, and hippocampus of animals with spontaneous seizures, without modification of Kd values. Such changes could be considered to be involved in some of the physiological and behaviour activities observed in this strain of rats.


Gastric small cell carcinoma with squamous and neuroendocrine differentiation. A rare gastric carcinoma containing diverse components, that is, neuroendocrine (small cell carcinoma), squamous and gland-like elements in an 82 yr old woman is described. Radiologic examination revealed a large ulcerated tumor, and a Borrmann type II tumor, 6.5 x 5 cm, was found in the resected stomach. Histologically, the tumor was mainly composed of small cells with hyperchromatic nuclei and scant cytoplasm. Argyrophilic granules were seen in these cells. There were also scattered foci of large cells with features of squamous cells, and many intermediate cells with oncocytic cytoplasm. The small cancer cells were positive for chromogranin A and neuron specific enolase. Squamous cell nests were positive for high molecular cytokeratin (CK), and intermediate cells were positive for low molecular CK. Electron microscopic examination revealed secretory granules in the small cells and tonofilaments in the squamous cells. This tumor might have originated from the pluripotential stem cell in the gastric epithelium.


Collision tumor: serous adenocarcinoma and steroid cell tumor of the ovary. A "collision" tumor between a serous papillary adenocarcinoma and a steroid cell tumor of the ovary is described. No similar combination has been reported in the literature. The steroid cell component secreted testosterone, resulted in considerable virilization of the patient, and appears to have preceded the carcinoma by several years. It remains problematical whether the androgenic milieu may have predisposed to the development of the second, malignant, tumor.


In situ hybridization at light and electron microscopic levels: identification of human papillomavirus nucleic acids. A comparison of the sensitivities of biotinylated and 32P-labelled human papillomavirus type 6b DNA probes was made. Slot blot hybridization results showed that the sensitivity of biotinylated probes was consistent with that of 32P-labelled, that is, 0.1 pg of pBR 322 plasmid containing 8 kbp HPV cDNA. In situ hybridization using 35S-labelled probes was applied to tissue from condylomata acuminata. After autoradiography, many silver grains were seen concentrated over the superficial koilocytic nuclei with some grains present in the cytoplasm. Biotinylated probes were visualized by 4 different means, i.e., streptavidin alkaline phosphatase, streptavidin biotinylated horseradish peroxidase, monoclonal anti-biotin antibody with 15 nm colloidal gold and streptavidin 5 nm colloidal gold. Strong reaction products were localized in the superficial nuclei while the cytoplasm of koilocytes showed weak hybridization signal. Pre-embedding methods were carried out for electron microscopic studies in which numerous granular diaminobenzidine (DAB) products were present in the nuclear chromatin while viral particles themselves had much fewer DAB products. This suggested that hybridization occurred more efficiently to yet unassembled viral genomes than to matured virions. Post-embedding methods using 15 nm colloidal gold were performed and showed singly scattered or clustered gold particles in superficial koilocytic nuclei.


Varicella in pediatric renal transplant recipients. As of November 1991, 8 of 83 children who had received renal transplants at Massachusetts General Hospital since January 1979 required admission for primary varicella. All 8 had cutaneous manifestations of disease, and 4 had evidence of visceral disease. Three of these 8 children received varicella zoster immune globulin (VZIG) after exposure to varicella; in the remaining children, exposure was not revealed until symptoms were present. All 8 children were treated with high-dose intravenous acyclovir. Two children died of complications of varicella infection, including 1 child who received VZIG on the day of exposure to varicella. Neither VZIG prophylaxis nor treatment with intravenous acyclovir offers complete protection against severe varicella infection to immunosuppressed children who have received organ transplants. A high priority should be given to the evaluation of alternative treatments, such as vaccination to the varicella virus, which could be administered to susceptible transplant candidates, preferably prior to transplantation.


[Clinical application of 31P MRS to malignant liver tumors--with an emphasis on evaluation of response to therapy]. Malignant liver tumors in 19 cases (13 hepatomas and 6 metastatic tumors) were studied by 31P magnetic resonance (MR) spectroscopy. Five healthy volunteers were also studied. Volume localization was performed using the ISIS sequence. Compared with normal liver, the MR spectra of malignant tumors showed an elevated phosphomonoester (PME) peak relative to beta ATP (p less than 0.005), and tumors were slightly alkaline (pH 7.32 +/- 0.08) before therapy. Nine cases of hepatomas responding to chemoembolization showed extremely reduced signal intensity of spectra. Tumor pH was compared before and after therapy in four cases that showed good response to therapy, and was elevated in all of them. Three metastatic tumors with good response showed reductions in PME and ATP after therapy. MR spectroscopy with adequate localization technique is useful in demonstrating the response of malignant tumors to therapy.


[Intrahepatic microvascular changes after intrahepatic arterial injection of lipiodol in normal and cirrhotic rats]. Iodized oil (Lipiodol) has been used for the diagnosis and treatment of hepatic malignancies, especially that of hepatocellular carcinoma. However, few reports have appeared concerning intrahepatic microvascular changes following intrahepatic arterial injection of Lipiodol. In the present study, Lipiodol was injected into the hepatic arteries of normal and cirrhotic rats, the changes of intrahepatic microvascular structures were serially investigated by scanning electron microscopy of microvasculature casts. In normal rats following intrahepatic arterial injection of Lipiodol, impairments of capillary vessels in the peribiliary plexus (PBP) were noted. The degrees of PBP impairments were most severe 2 days after the injection of Lipiodol. However, 10 days after the injection of Lipiodol, these impairments had almost completely vanished. There was no changes of terminal arterial and portal branches. In cirrhotic rats, proliferations of the perinodular arterial plexus (PNAP) and dilatations of the PBP were noted. After intrahepatic arterial injection of Lipiodol, the impairments of the PBP were noted, as in the normal rats. However, the degrees of these impairments were milder than those in the normal rats. It was considered that the decrease of the PBP impairments caused by Lipiodol were influenced by proliferation of the PNAP and dilatations of the PBP in cirrhotic rats. After intrahepatic arterial injection of Lipiodol in normal and cirrhotic rats, there was no development of collateral vessels. It was believed, therefore, that this fact was an advantage in the treatment of hepatic carcinoma by Lipiodol.