[Susceptibility of RD-9H8 cells derived from RD cells to enteroviruses and its application for measuring neutralizing antibodies against coxsackievirus group B]. We carried out cloning of RD cells and obtained cloned cell line RD-9H8, which is highly sensitive to enteroviruses. Cytopathic effect (CPE) of RD-9H8 cells was more remarkable than that of original RD cells. The RD-9H8 cells were used to measure the neutralizing antibodies to coxsackievirus group B in sera from 93 medical students collected in Oct. 1990 at Iwate Medical University. The proportions of confirmed positive sera were coxsackievirus type B1 44.1%, type B2 50.5%, type B3 46.2%, type B4 64.5%, type B5 53.8% and type B6 4.4%.


Cleft hand, syndactyly and hypoplastic thumb. The clinical features of 58 patients with typical cleft hand were examined and compared with 86 patients with syndactyly between the long and ring fingers, 27 patients with syndactyly between the ring, little and other fingers, 53 patients with hypoplastic thumb and 100 patients with symbrachydactyly. The clinical findings of the typical cleft hand resembled those of syndactyly. There were two unusual cases of typical cleft hand associated with hypoplastic thumb, congenital heart anomalies and absence of the axial triradius. One of these also had cleft lip and palate. The critical embryonic period of the heart anomaly is early, while that of the cleft lip and palate is late. These findings suggest that an embryo with typical cleft hand and hypoplastic thumb results from impairments at two different times in the early embryonic period.


Multiple glomus tumours.

Multiple glomus tumours. A patient with multiple, synchronous, non-familial head and neck paragangliomas is reported. There were three primary neoplasms, a glomus tympanicum and glomus vagale on the right side and a glomus tumour of the carotid body on the left. Such a combination has never been reported previously. The reports of all the series with paragangliomas in the literature, as well as the reports of single cases with multiple tumours during the last three decades, are reviewed. Specific problems in diagnosis and management of multiple glomus tumours are discussed.


Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins. Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.


Synthesis, characterization, cytotoxicity and DNA binding studies of diamminediethyldithiocarbamato-platinum(II) nitrate. A new complex [Pt(NH3)2(ddtc)]NO3.2H2O as a 1:1 electrolyte has been prepared. This was characterized by spectroscopic methods. The electronic absorption spectrum of this complex in water suggests that it has a square planar geometry. The infrared, 1H NMR and x-ray photoelectron spectroscopic studies suggest the bonding of ammonia molecules and diethyldithiocarbamate as bidentate ligand to platinum(II) in this complex. The 50% inhibition value of this complex against P388 lymphocytic leukemia cells is comparable with cisplatin. This complex interacts with calf thymus DNA by coordinate covalent bond.


The role of dianion coupling in the synthesis of dibenzylbutane lignans. Dianion coupling reactions have been used to prepare structurally related lignan natural products in racemic form. A readily available alpha-idocarboxylic acid 10 has been employed in separate sequences to afford the tetrahydrofuran lignan burseran [6] and the butyrolactone lignan deoxypodorhizon [7]. The key strategy for both burseran and deoxypodorhizon involved reaction of the appropriate dianion with an alpha-iodo carboxylate salt 13. Equilibration under acid or base was employed to create the trans stereochemistry of the substituted benzyl side chains of both burseran and deoxypodorhizon. To achieve proper dianion reactivity and to avoid side reactions in the synthetic sequence to deoxypodorhizon, it was necessary to develop the chemistry of acylsulfonamide dianions. As a further structure proof, our synthetic deoxypodorhizon was also utilized as a substrate in a successful sequence to isostegane [8], a known relay intermediate to the antineoplastic prototype steganacin [9].


Defects of the mitochondrial respiratory chain complexes in three pediatric cases with hypotonia and cardiac involvement. Three children displaying hypotonia, cardiac involvement and defects of the mitochondrial respiratory chain complexes are reported. The first case showed severe neonatal hypotonia, failure to thrive, hepatomegaly, dilation of the right cardiac cavities, profound lactic acidosis and amino aciduria. The boy died at the age of 7 weeks. In the second case hypotonia, severe cardiomyopathy, cyclic neutropenia, lactic acidosis and 3-methylglutaconic aciduria occurred. The boy died at the age of 27 months. The third case presented at the age of 16 months as an acute hypokinetic hypertrophic cardiomyopathy with transient hypotonia and mild lactic acidosis. Spontaneous clinical remission occurred. In all cases muscle biopsy was performed. Morphological studies failed to show ragged-red fibers but there was lipid storage myopathy and decreased cytochrome c oxidase activity. Biochemical studies confirmed the cytochrome c oxidase deficiency in muscle in all cases. It was associated with complex I III deficiency in case 1 and with severe deficits of all respiratory chain complexes in case 2. Post-mortem studies in case 1 indicated that complex IV was reduced in the liver but not in the heart and quantitative analysis of mtDNA revealed a depletion in muscle. Cases 1 and 2 shared some clinical features with fatal infantile myopathy associated with cytochrome c oxidase deficiency, while case 3 displayed a very unusual clinical presentation. The histochemical enzyme reaction of cytochrome c oxidase is useful for the diagnosis of mitochondrial myopathy because ragged-red fibers may be lacking. Finally, biochemical measurement of the different mitochondrial respiratory chain complexes is required because multiple defects are frequent and occasionally related to mtDNA depletion.


Familial motor neuron disease with Lewy body-like inclusions in the substantia nigra, the subthalamic nucleus, and the globus pallidus. In a familial case of motor neuron disease (MND), 2 unusual features were noted in the necropsy. The first was a pallidoluysonigral degeneration, observed in only 4 other cases of MND and which was here asymptomatic. The second was the presence in degenerated spinal cord anterior horns and in degenerated basal ganglia of neuronal Lewy body-like inclusions stained by antibodies against ubiquitin.


Changes of neuropeptides and their receptors in experimental stroke gerbil brains. Eight kinds of neuropeptides and four kinds of neuropeptide receptors were examined in the right and left hemispheres of mongolian gerbils after unilateral carotid ligation-induced stroke and in normal controls. Five hours after ligation of the right common carotid artery, beta-endorphin concentration in the right hemisphere (ischemic side) of the stroke group was significantly increased compared with that in the contralateral hemisphere (non-ischemic side), but there were no differences between sides in other neuropeptides either with or without stroke. Furthermore, although there were no differences in [3H]naloxone binding, [3H]thyrotropin-releasing hormone binding or 125I-vasoactive intestinal polypeptide binding in the brain in this model of stroke, [3H]enkephalin binding was significantly lower on the ischemic side than on the non-ischemic side in the stroke group. These results suggest that increased activity in the beta-endorphinergic system in the brain might be partly caused by ischemic brain failure.


A pseudoreceptor docking study of 4,5-alpha-epoxymorphinans with a range of dielectric constants. Thirteen 4,5-epoxymorphinan mu agonists with established analgesic action were docked into an Asp-Lys-His-Phe pseudoreceptor complex under a range of distance-dependent dielectric conditions. The number of compounds with potential energies of the docked complexes that agreed in rank order with corresponding analgesic potencies was determined for each condition. Two dielectric conditions, n-decane (1.991) and ethanol (24.3), enabled the greatest number of compounds to relate to their pseudoreceptors with each having 9 and 8 successes respectively. Both of these conditions demonstrated unique influences on the types of structures that were successfully docked. For example, the morphine stereoisomer alpha-isomorphine, the geometric isomer B/C trans-morphine, and the 8-position-substituted gamma-isomorphine were successes in the n-decane condition, whereas the ethanol condition produced the substituted codeine derivatives dihydrocodeinone and dihydroxycodeinone. These findings emphasize the importance of dielectric influence when developing force-field modeled quantitative structure-activity relationships for a closely related homologous series.


Association of epidermal growth factor receptor gene amplification with loss of chromosome 10 in human glioblastoma multiforme. Although the loss of tumor suppressor genes and the activation of oncogenes have been established as two of the fundamental mechanisms of tumorigenesis in human cancer, little is known about the possible interactions between these two mechanisms. Loss of genetic material on chromosome 10 and amplification of the epidermal growth factor receptor (EGFR) gene are the most frequently reported genetic abnormalities in glioblastoma multiforme. In order to examine a possible correlation between these two genetic aberrations, the authors studied 106 gliomas (58 glioblastomas, 14 anaplastic astrocytomas, five astrocytomas, nine pilocytic astrocytomas, seven mixed gliomas, six oligodendrogliomas, two ependymomas, one subependymoma, one subependymal giant-cell astrocytoma, and three gangliogliomas) with Southern blot analysis for loss of heterozygosity on both arms of chromosome 10 and for amplification of the EGFR gene. Both the loss of genetic material on chromosome 10 and EGFR gene amplification were restricted to the glioblastomas. Of the 58 glioblastoma patients, 72% showed loss of chromosome 10 and 38% showed EGFR gene amplification. The remaining 28% had neither loss of chromosome 10 nor EGFR gene amplification. Without exception, the glioblastomas that exhibited EGFR gene amplification had also lost genetic material on chromosome 10 (p less than 0.001). This invariable association suggests a relationship between the two genetic events. Moreover, the presence of 15 cases of glioblastoma with loss of chromosome 10 but without EGFR gene amplification may further imply that the loss of a tumor suppressor gene (or genes) on chromosome 10 precedes EGFR gene amplification in glioblastoma tumorigenesis.


Papilloma virus in cervical carcinoma: detection of viral antigen in cancer cells. Paraffin sections of biopsies from histopathologically confirmed cases of uterine cervical carcinoma, cervical scrapings from dysplasia, chronic cervicitis, tumour cells from carcinoma of the oral cavity and normal tissues from healthy normal cervix and oral cavity scrapings were examined for the presence of Human papilloma virus antigens. The techniques adopted were the Indirect Immunofluorescence Stainig and the Peroxide-Anti-Peroxidase techniques. The HPV-antigen was present in 38 percent and 41 percent of invasive carcinoma cervix, by PAP and IIF methods respectively. In cervical dysplasia 8-13% revealed HPV antigen while oral carcinoma cells and normal tissue samples were totally negative.


Detection of herpes simplex virus type-2 DNA and human papilloma virus DNA sequences in cervical carcinoma tissue by molecular hybridization. Biopsy samples from one hundred and two patients with squamous cell carcinoma of the uterine cervix and tissues from twelve healthy normal cervical tissues (post hysterectomy) were examined for HSV-type 2 and HPV-11 DNA sequences by molecular hybridization technique. In the carcinoma tissue extracts 53% contained HSV-2 DNA, 27% -HSV-1-DNA and 36% showed HPV-11 gene sequences while 5.7% were found to contain both HPV and HSV-2 DNA. Biopsies from healthy cervix were completely negative for HSV-2 and HPV-11 DNA sequences.


Anti-HHV-6 antibodies in normal population and in cancer patients in India. The prevalence and titre of IgG antibodies to human herpesvirus type 6 (HHV-6) were assayed in the serum samples from normal subjects and patients with Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukaemia (ALL) and oral cancer (OC) using immunofluorescence and immunoperoxidase techniques. This forms the first study on the sero-prevalence and titre of antibodies to HHV-6 in India. There was no considerable difference in the prevalence (76%) and titre (10-160) of the antibodies in normal population from those reported for normal adults in other parts of the world. All the HL and ALL patients studied showed no significant elevation in the antibody titre, though a slight increase in the prevalence (95%) was noted. Antibody titre and prevalence were found highly elevated in OC. OC remained totally unstudied for the presence of anti-HHV-6 antibodies, and this is the first report of elevated levels of the antibody in this cancer. The role of HHV-6, if any, in the pathogenesis of OC is worth investigating.


Immunoreactivity for the beta chain of the platelet-derived growth factor receptor in malignant mesothelioma and non-neoplastic mesothelium. The results of an immunohistochemical study of platelet-derived growth factor receptor (PDGF-R) beta chain in human non-neoplastic mesothelium (35 cases) and in malignant mesothelioma (33 cases) using a murine monoclonal antibody are reported. In approximately half of the malignant mesotheliomas, there was immunoreactivity in the cytoplasm of the neoplastic cells for the beta chain of the PDGF-R. In some cases, there was also membrane immunoreactivity. There were no statistically significant differences between the various mesothelioma subtypes. Immunoreactivity for PDGF-R beta chain was absent in non-neoplastic mesothelium.


Recombinant human interferon gamma therapy for osteopetrosis. A defect in leukocytic superoxide formation has been demonstrated in patients with congenital osteopetrosis. This leukocyte defect appears to be related to defective bone resorption. Because recombinant human interferon gamma therapy enhances superoxide production in patients with chronic granulomatous disease, we sought to determine whether a similar strategy could reverse the osteopetrotic condition. Interferon gamma, 1.5 micrograms/kg three times a week, was administered by subcutaneous injection for 6 months to eight patients with osteopetrosis. Urinary hydroxyproline and urinary calcium excretion increased markedly during therapy in parallel with a significant decrease in trabecular bone volume. Bone marrow scans demonstrated increased bone marrow production. The hemoglobin concentration, platelet count, and leukocyte production of superoxide increased significantly. No serious infections were encountered during the therapy. These data suggest that interferon gamma administration enhances bone resorption and leukocyte function in patients with osteopetrosis.


Chronic hepatitis B surface antigen-negative hepatitis after treatment of malignancy. We reviewed the records of all patients with a diagnosis of malignancy who were treated at our center and who had not had chemotherapy for at least 18 months, to assess the prevalence of chronic hepatitis B surface antigen (HBsAg)-negative hepatitis, to assess the prevalence of a marker of hepatitis C virus infection, and to determine the severity of chronic liver disease. Of 557 eligible patients, 38 (6.8%) had chronic HBsAg-negative hepatitis. Of these 38 patients, 20 (52.6%) had a marker of hepatitis C virus infection. The prevalence of chronic HBsAg-negative hepatitis was higher in patients previously treated for leukemia than in patients treated for another malignancy (11.8% vs 4.6%; p = 0.004). The liver biopsy revealed chronic active hepatitis or cirrhosis or both in 8 (28%) of 28 patients with clinical chronic HBsAg-negative hepatitis. Four patients without hepatitis C virus infection who underwent liver biopsy had hepatitis B virus antigen in the liver, confirmed by immunohistochemistry studies. One patient uninfected with hepatitis C virus had hemochromatosis. We conclude that infection with hepatitis C virus was the major cause of chronic HBsAg-negative hepatitis in pediatric patients previously treated for malignancy; the cause remained unidentified in 30% of the patients.


Superior mesenteric artery injury during nephrectomy for Wilms' tumor. Iatrogenic injury to the aorta or its major branches during nephrectomy for Wilms' tumor in children is rarely reported but may be more common than is currently acknowledged. We identified four patients with ligation of the superior mesenteric artery (SMA) that occurred during nephrectomy for nephroblastoma and another child in whom SMA thrombosis developed postoperatively. All of the tumors were on the left side. Interruption of the SMA was recognized intraoperatively in all four children, and primary repair was done. In three patients, appearance of the bowel remained normal before repair of the injury. Three of the arteries were repaired by primary reanastomosis, and one was joined with an interpositioned hypogastric artery graft. None of these patients had gastrointestinal complications postoperatively. In the fifth patient, SMA thrombosis developed after repair of an aortic tear during nephrectomy. This patient required subsequent small bowel resection for bowel infarction and died in the perioperative period. Every surgeon treating children with Wilms' tumor should be aware of the possibly distorted vascular anatomy and take precautions to avoid such a significant injury. Attempts at early ligation of the vessels may not be justified until the renal vasculature is clearly identified.


Benign testicular tumors in children with congenital adrenal hyperplasia. The association between testicular tumors/nodules and congenital adrenal hyperplasia (CAH) has been previously reported. From 1960 to 1989, three patients (13 to 18 years old) with long-standing CAH developed testicular masses. Two patients with 21-hydroxylase deficiency were diagnosed in the neonatal period while one other with 11-hydroxylase deficiency was diagnosed at 3 years of age when he presented with sexual precocity. In all three patients, medical compliance was poor. The testicular masses were bilateral in two patients and unilateral in one, measured 1 to 2 cm, and occupied only the upper half of the testicle. Testicular biopsy specimens were obtained after at least 6 months of evidence of compliance with the adrenocorticotrophic hormone (ACTH) suppressive medication and failure of the nodules to regress. On gross examination the masses appeared to be firm yellow brown nodules. Light microscopy showed interlacing strands, cords, and rests of cells resembling interstitial (Leydig) cells but with no Reinke crystalloids. Electronmicroscopy in all patients showed variable amounts of both smooth and rough endoplasmic reticulum, the later with occasional dilated cisternae. Follow-up ranged from 6 months to 6 years. No further surgical treatment has been necessary. There has been no evidence of recurrence, distant metastases, or secondary malignancies during the time of follow-up. These findings suggest that testicular tumors may develop from chronic excessive ACTH stimulation of a putative pluripotential testicular cell, a Leydig cell, or an adrenal cortical rest. Unlike other testicular tumors these do not require orchiectomy as the initial form of therapy.


Wilms' tumor: a new cause for urinary extravasation. We report a case of Wilms' tumor associated with urinary extravasation due to tumor invasion through the renal pelvis and anterior renal capsule. Extravasation of urine exposed to tumor may lead to upstaging of the tumor and the requirement for more intensive therapy.


Detection of herpes simplex virus in gingival tissue. The presence of herpes simplex virus (HSV) antigens was shown by immunofluorescence staining in 26 of 66 (39.3%) specimens of clinically healthy gingiva, but only one sample contained infectious virus. HSV DNA sequences were clearly identified in intact gingival cells by dot blot hybridization in one specimen, and a weak pattern in a second one. Both specimens harbored viral antigens. These findings of viral genome and protein expression suggest that the virus is present in the latent form in the gingiva.


Effects of mushroom toxins on glycogenolysis; comparison of toxicity of phalloidin, alpha-amanitin and DL-propargylglycine in isolated rat hepatocytes. The effects of phalloidin and alpha-amanitin as toxins of Amanita species and DL-propargylglycine identified from A. abrupta on the glycogenolysis in isolated rat hepatocytes were investigated. Phalloidin decreased glycogen content and activated phosphorylase a activity remarkably. alpha-Amanitin also decreased glycogen content significantly but activated phosphorylase a activity slightly. DL-Propargylglycine slightly affected glycogenolysis. Phalloidin, which most affected glycogenolysis among the three compounds mentioned above, elevated cytosolic Ca2+ concentration ([Ca2+]i) and 45Ca uptake into cells. Phalloidin depressed slightly 3H-inositol incorporation into phosphatidylinositol (PI) and remarkably phosphatidylinositol 4,5-bisphosphate (PIP2) but increased phosphoinositides breakdown. These results suggest that phalloidin alters phosphoinositides turnover and intracellular Ca2+ homeostasis, subsequently activates phosphorylase a, resulting in glycogenolysis in isolated rat hepatocytes.


Antinociceptive activity of intrathecally administered cannabinoids alone, and in combination with morphine, in mice. The antinociceptive effects of various cannabinoids, alone and in combination with opiates, were evaluated in antinociceptive tests in mice. The cannabinoids tested produce marked antinociceptive effects after i.t. administration to mice. The rank order of potency for the drugs using the tail-flick test was levonantradol greater than CP-55,940 = CP-56,667 greater than 11-hydroxy-delta 9-THC greater than delta 9-THC greater than delta 8-THC; dextronantradol was inactive at a dose of 25 micrograms/mouse. Respective ED50 values in the tail-flick test were 0.4, 12.3, 4.2, 15, 45 and 72 micrograms/mouse. Although pretreatment with morphine somewhat enhanced the effects of delta 9-THC, pretreatment of the mice with naloxone (1 mg/kg s.c. or 1 micrograms/mouse i.t.) failed to block the antinociceptive effects of the cannabinoids, indicating that the cannabinoid-induced antinociception does not occur due to direct interaction with the opiate receptor. Pretreatment of mice with 3.13 micrograms/mouse and 6.25 micrograms/mouse of delta 9-THC shifted the ED50 of morphine to 0.15 and 0.05 micrograms/mouse, respectively (a 4-and a 12-fold shift). The shifts in the dose-response curve of the morphine were parallel. Naloxone administration (1 mg/kg s.c.) completely blocked the antinociceptive effects of the combination of 6.25 micrograms of delta 9-THC with morphine. The AD50 for naloxone blockade of the drug combination was 0.24 (0.06-0.94) mg/kg s.c. and the pA2 was 7.7 (6.7-8.9). The pA2 for naloxone blockade of the dimethylsulfoxide-morphine combination was 6.9 (5.7-8.1).(ABSTRACT TRUNCATED AT 250 WORDS)


Handling of digoxin and ouabain by renal tubular cells (LLC-PK1). Digoxin is known to be secreted by renal tubular cells, but the mechanisms are still not fully understood. In this study, we examined renal tubular cell handling of digoxin and ouabain using LLC-PK1 cells, a model of proximal renal tubular cells. The cells were used in suspension for binding experiments and in monolayers on permeable filters for transport studies. The specific binding of digoxin to the cells, presumably to the ouabain binding site (i.e., membrane Na+,K(+)-ATPase), were characterized by Kd of 2.6 x 10(-7) M and Bmax (total number of specific binding sites) of 1.6 x 10(6)/cell. Kd and Bmax of ouabain binding were 1.3 x 10(-7) M and 1.9 x 10(6)/cell, respectively. In transport experiments, digoxin showed significantly higher flux than ouabain from the basolateral to the apical side across the cell monolayers. Importantly, this secretory transport was not inhibited by ouabain concentrations sufficient to block membrane Na+,K(+)-ATPase and to displace digoxin from the binding site on the enzyme (i.e., 10(-6) to 10(-4) M ouabain). However, the digoxin secretion was decreased by low temperature or excess digoxin in a concentration-dependent manner. These data suggest that digoxin undergoes unidirectional transport in favor of secretion, which does not involve its binding to the ouabain binding sites on membrane Na+,K(+)-ATPase.


Mediation of swim-stress antinociception by the opioid delta 2 receptor in the mouse. The present study has characterized the antinociceptive response to cold water swim-stress (CWSS) in mice using opioid-selective antagonists as well as tolerance and cross-tolerance approaches. Mice subjected to CWSS using water at 5 degrees C for 3 min showed a marked antinociceptive response in the tail-flick test, which reached approximately 90% after +10 min, and which persisted for 15 to 20 min. This antinociceptive response (at +10 min) was antagonized by naloxone or by the delta antagonist ICI 174,864. Additionally, the CWSS response was antagonized by the opioid delta 2 antagonist, naltrindole-5'-isothiocyanate, but not by the delta 1 antagonist, [D-Ala2,Leu5,Cys6]enkephalin, or by the mu antagonist, beta-funaltrexamine or by the kappa antagonist, norbinaltorphimine. Although the CWSS-induced antinociceptive effect was blocked by some delta antagonists and tolerance resulted from the CWSS-induced response, the decrease in body temperature after each CWSS exposure was not affected by the opioid antagonists and reliably occurred in CWSS-tolerant mice, suggesting that the observed antinociception was independent of changes in body temperature. In mice rendered tolerant to the antinociceptive actions of the mu agonist, [D-Ala2,NMPhe4,Gly-ol] enkephalin, or to [D-Pen2,D-Pen5]enkephalin (predominantly a delta 1 agonist), the CWSS-induced antinociceptive response was unaltered. In contrast, in mice tolerant to the delta 2 agonist, [D-Ala2,Glu4]deltorphin, the CWSS-induced antinociceptive response was markedly and significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)


Reduction in potency of selective gamma-aminobutyric acidA agonists and diazepam in CA1 region of in vitro hippocampal slices from chronic flurazepam-treated rats. The potency and efficacy of selective gamma-aminobutyric acidA (GABAA) agonists (GABA, muscimol, isoguvacine and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol), the GABAB agonist, baclofen, and the benzodiazepine agonist, diazepam, were examined using extracellular recording techniques in in vitro hippocampal slices from rats sacrificed 2 days after 1 week of flurazepam treatment. Population spikes elicited by stimulation of Schaffer collaterals were recorded in the CA1 pyramidal cell region with NaCl-containing glass micropipettes. GABA agonists were superfused in increasing concentrations for 5 min. Drug responses, averaged over the last 2 min for each concentration, were compared to the predrug base line. GABAA agonists, but not baclofen, showed a significant, 2-fold, decrease in potency, but not efficacy, to reduce CA1-evoked responses in treated vs. control slices. The benzodiazepine effect was evaluated by the shift in the isoguvacine dose-response curve in the absence, then presence, of diazepam. A reduction in diazepam potency was demonstrated in vitro by a significantly reduced shift in the isoguvacine curve by 300 nM, but not 1 microM, diazepam after chronic but not acute in vivo pretreatment. The results indicated a selective GABAA agonist subsensitivity and diazepam tolerance in hippocampus after 1 week of flurazepam treatment and establish the hippocampal slice preparation as a valuable substrate for investigating synaptic mechanisms of benzodiazepine tolerance.


Lisinopril and ramiprilat protection of the vascular endothelium against free radical-induced functional injury. We reported earlier that the vasodilator response to acetylcholine (ACh) in lungs exposed to indomethacin and preconstricted with an analog of thromboxane (U46619) is converted to vasoconstriction by brief electrolysis of inflowing perfusion medium and suggested that this effect reflected endothelial injury. The purpose of our present study was 2-fold. First, because captopril, a sulfhydryl-containing inhibitor of angiotensin-converting enzyme inhibitor, prevented this effect (we assumed by scavenging electrolysis generated free radicals of oxygen), we determined whether two angiotensin-converting enzyme inhibitors lacking this moiety, namely lisinopril and ramiprilat, provided similar protection. Second, we studied whether electrolysis, like other forms of experimental lung injury, impaired uptake of serotonin (5-HT) by the endothelium. Our study confirmed that within 5 min of electrolytic injury, the ACh response is converted to vasoconstriction. This effect was completely prevented by lisinopril (18 microM) or ramiprilat (30 microM), neither of which affected ACh vasodilatation in control lungs. Lower concentrations of either drug exerted lesser degrees of protection. Five or 20 min after electrolysis, single-pass uptake of [14C]5-HT was significantly (P less than .01; N = 11) lower than control (82.4 +/- 3.4% vs. 71 +/- 3.2 and 46.5 +/- 6%, respectively). In contrast, 5-HT uptake was unaltered by electrolysis in the presence of 18 microM lisinopril. We conclude that loss of ACh vasodilation is an early reflection of lung endothelial injury that is accompanied by reduced [14C]5-HT uptake. Also, the protective property of nonsulfhydryl-containing angiotensin-converting enzyme inhibitors may be related to unexpected antioxidant actions.


Further evidence that morphine-6 beta-glucuronide is a more potent opioid agonist than morphine. The antinociceptive properties of morphine-6 beta-glucuronide (M6G) and morphine (oral, i.c.v. and s.c.) were examined in two tests involving different nociceptive stimuli [i.e., cutaneous-thermal (tail-flick) and chemical-visceral (acetic acid-writhing)] in both naive and chronically treated mice. Twenty min after i.c.v. injection, M6G was 47 and 360 times more potent than morphine in the writhing and tail-flick tests, respectively. This difference was not due to differences in affinity because M6G displayed lower apparent affinities (Ki) for mu and kappa binding sites in vitro. After systemic injection, the two opiates were equieffective, although M6G produced a 10-fold longer antinociceptive effect. These differences with route of administration partially result from the hydrophilic nature of M6G because its inflow into the brain compartment was at least 10-fold lower than that of morphine, whereas the rate of elimination of the parent molecule was 3 times greater. After chronic treatment, mice readily develop tolerance and marked physical dependence to the antinociceptive effects of M6G. In vivo binding studies showed that M6G exerts its antinociceptive effect at low (less than 1%) fractional occupancy of [3H]diprenorphine-specific binding sites. In contrast, morphine needs to occupy 9.5 (writhing) to 47 (tail-flick) times more opioid binding sites to produce the same antinociceptive activity. M6G thus appears to have greater pharmacological potency than morphine, which in comparison possesses a low intrinsic efficacy.


Activation of protein kinase A is necessary but not sufficient for ethanol-induced desensitization of cyclic AMP production. Acute addition of EtOH to PC 12 pheochromocytoma cells increases cyclic AMP production, whereas chronic exposure to EtOH results in a decrease in the stimulation of cyclic AMP production in response to 2-chloroadenosine and forskolin. This EtOH-induced desensitization was not observed after chronic EtOH treatment of A126-1B2-1 cells which are a protein kinase A-deficient mutant cell line derived from PC 12 cells. Furthermore, in the parental PC 12 cell line the cell-permeable protein kinase A inhibitor, Rp-isomer of adenosine 3',5'-monophosphorothioate, blocked the development of EtOH-induced desensitization. Thus, activation of protein kinase A is apparently necessary for EtOH-induced desensitization of cyclic AMP production. Chronic treatment of PC 12 cells with forskolin qualitatively mimicked the desensitization observed with chronic EtOH exposure. However, the degree of desensitization induced by forskolin was significantly less than that caused by EtOH even though the acute addition of forskolin caused a greater increase in cyclic AMP production. Furthermore, the acute addition of EtOH inhibited forskolin-stimulated cyclic AMP production, yet inclusion of EtOH during the chronic forskolin treatment of PC 12 cells resulted in a greater degree of desensitization. These findings indicate an obligatory role of protein kinase A in EtOH-induced desensitization of cyclic AMP production in PC 12 cells. However, because protein kinase A activation alone is not sufficient to account for the degree of desensitization, EtOH probably also acts through a mechanism in addition to activation of protein kinase A.


Age-associated enhancement of diquat-induced lipid peroxidation and cytotoxicity in isolated rat hepatocytes. The effect of age on the toxicity of diquat, a redox cycling compound, was investigated in hepatocytes isolated from mature (6 months) and old (24-29 months) male Fischer 344 rats. Hepatocytes of old rats were more sensitive than those of mature rats to diquat-induced cytotoxicity (lactate dehydrogenase release into the medium). Cell death was preceded by glutathione disappearance, and rates of glutathione depletion were similar in mature and old hepatocytes. In contrast, diquat-induced formation of thiobarbituric acid-reactive substances was much greater in the hepatocytes from old rats, suggesting that increased lipid peroxidation caused the enhanced cytotoxicity. Further experiments revealed that: 1) hepatocytes of mature and old rats were equally sensitive to iron-induced lipid peroxidation; 2) diquat-stimulated production of superoxide anion radical in liver microsomes did not increase with age, but decreased 43%; 3) superoxide dismutase activity was similar in hepatocytes of mature and old rats; 4) inhibition of catalase activity (which diminishes with age in male rats) did not increase diquat toxicity; and 5) malondialdehyde disappearance in intact hepatocytes decreased (33%) with age, but the toxicological significance of the decline in metabolism was uncertain. Thus, the results demonstrated that diquat-induced lipid peroxidation and cytotoxicity increase with age in male rat hepatocytes, but the enhanced sensitivity to diquat poisoning remains unexplained.


Identification and characterization of delta opioid binding sites in the bovine pineal. Bovine pineal membranes were shown to possess a single class of high-affinity binding sites for the opioid peptide, [125I]iodiotyrosyl27-beta-endorphin (beta E) (Kd = 47 pM, Bmax = 2.4 fmol/mg of tissue). The rank order of potency at this beta E site was deltorphin greater than [D-Ser2]-Leu-enkephalin-Thr greater than [D-Pen2,D-Pen5]enkephalin much greater than dermorphin greater than [D-Ala2,MePhe4,Gly5-ol]enkephalin much greater than (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl]] benzeneacetamide (U69593) greater than [des-Tyr1]beta E greater than beta E(6-31). These results suggest that beta E binds to delta opioid sites and excludes the possibility of significant binding to mu, kappa and epsilon sites. The presence of delta binding sites was confirmed by use of the delta selective ligand [3H][D-Pen2,D-Pen5]enkephalin (Kd = 1.5 nM). The Bmax observed using [D-Pen2,D-Pen5]enkephalin is similar to that obtained with [125I]beta E, confirming that essentially all pineal opioid sites are of the delta type. The virtual absence of mu opioid sites was confirmed using the mu-selective opioid ligand [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin. These results suggest that endogenous or circulating opioid peptides may modulate pineal function by interaction with delta opioid sites.


Continuous intrathecal opioid treatment abolishes the regulatory effects of magnesium and guanine nucleotides on mu opioid receptor binding in rat spinal membranes. The regulatory effects of cations and guanine nucleotides on mu receptor binding after opioid drug and pertussis toxin treatment were studied in the rat spinal cord model. Continuous intrathecal (i.t.) infusion with PL017 for 5 days induced tolerance in a dose-dependent manner. Maximal tolerance was observed at day 2. A single i.t. dose (1 microgram) of pertussis toxin also induced tolerance to opioid. When mu receptor binding of the high-affinity sites was determined by 125I-FK33824, spinal membrane preparations from morphine- and pertussis toxin-induced tolerant animals demonstrated approximately 30% less binding than control membranes. Analysis of equilibrium competition binding of FK33824 against [3H]naloxone under a variety of experimental conditions (i.e., cations and guanine nucleotides) revealed differences among control and treated membranes. With Na+ (100 mM) + GDP (100 microM) pretreated membranes and binding assays conducted in the presence of Mg++, all mu receptors were observed to be in a high-affinity state in control membranes, whereas about 30% of receptors were in the low-affinity state in membranes from opioid- and pertussis toxin-treated animals. The increase in the proportion of low-affinity sites was dependent upon the infusion dose of PL017, and the increase correlated well with the degree of opioid tolerance developed. The regulatory effect of 5'-guanylylimidodiphosphate on opioid agonist binding was reduced in membranes from pertussis toxin- or opioid-treated animals. In binding assays conducted in the presence of Na+ (100 mM) + Mg++ (5 mM) + 5'-guanylylimidodiphosphate (30 microM) or Na+ (100 mM) + GDP (100 microM), all mu receptors in control membranes were in a low affinity-state, while those from opioid- or pertussis toxin-treated animals existed in both the high- and the low-affinity states. Continuous i.t. infusion with PL017 at the high dose of 1 microgram/hr for 5 days also decreased significantly (about 40%) the total number of receptors. These studies indicate that continuous opioid infusion and pertussis toxin treatment results in impairment in the receptor-G-protein coupling. This is reflected by the decreased regulatory effects of Mg++ and guanine nucleotides. Thus, in addition to receptor down-regulation, which is induced by PL017 at high doses, receptor-G-protein uncoupling may play a role in opioid tolerance induced by continuous infusion with morphine and PL017.


Characterization of the enantiomers of cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine (BD737 and BD738): novel compounds with high affinity, selectivity and biological efficacy at sigma receptors. A novel class of compounds with very high affinity and selectivity for sigma receptors has been discovered. BD614 [(+/-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine] and its optically pure 1S,2R-(-)-[BD737] and 1R,2S-(+)-[BD738]enantiomers bound to sigma receptors of guinea pig brain with Ki = 2.0 +/- 0.4, 1.3 +/- 0.3 and 6 +/- 3 nM, respectively. These compounds exhibited little or no affinity for dopamine-D2, kappa opiate or phencyclidine receptors and displayed high biological efficacy in assays of sigma receptor function, ability to produce alterations in motor behavior and inhibition of the muscarinic cholinergic phosphoinositide response. Microinjection of BD614 into the rat red nucleus or substantia nigra produced a dose-dependent alteration in head position and contralateral circling, respectively. BD614, BD737 and BD738 inhibited stimulation of inositol phosphate production by carbachol or oxotremorine-M in a dose-dependent manner. Thus, N-substituted cis-2-(1-pyrrolidinyl)cyclohexylamines may prove useful in studies of sigma receptor structure and function.


Disulfiram and diethyldithiocarbamate are competitive inhibitors at the peripheral benzodiazepine receptor. In the present study in vitro interactions of disulfiram (an agent used to induce ethanol intolerance in alcoholics), diethyldithiocarbamate (DDC), and metronidazole with central benzodiazepine receptors (CBR) and peripheral benzodiazepine (BZ) receptors (PBR) were investigated in rat tissues. Disulfiram displaced specific binding of [3H]PK 11195 from PBR in the cerebral cortex with an IC50 value of 5 x 10(-7) M. The binding of [3H]PK 11195 and [3H]Ro 5-4864 to PBR in the kidney was displaced by disulfiram with IC50 values of 7 x 10(-7) and 2 x 10(-7) M, respectively. DDC displaced [3H]PK 11195 binding to kidney membranes with an IC50 value of 5 x 10(-5) M. Binding of [3H] flunitrazepam to CBR in the cerebral cortex was not affected by either disulfiram or DDC. Metronidazole (up to 10(-4) M), a disulfiram congener, did not affect [3H]flunitrazepam (FNZ) and [3H] PK 11195 binding to CBR and PBR, respectively. Scatchard analysis of [3H]PK 11195 binding to kidney membranes, performed in the absence or presence of 7 x 10(-7) M disulfiram, decreased ligand affinity without influencing the maximal number of binding sites, suggesting a competitive inhibition. Beta-Mercaptoethanol (2 x 10(-2) M), which blocks the inhibitory activity of disulfiram and DDC at the acetaldehyde dehydrogenase, did not affect the inhibitory potency of disulfiram at the kidney PBR. Removal of disulfiram from kidney by repeated washing with Tris-HCl buffer resulted in the restoration of binding properties to control values, suggesting reversibility of disulfiram binding to PBR.


Comparison of antagonist and agonist binding to the leukotriene B4 receptor intact human polymorphonuclear neutrophils (PMN). In the present studies, the pharmacology of the leukotriene B4 (LTB4) receptor on intact human polymorphonuclear neutrophils (PMN) was characterized using radioligand binding techniques with [3H]LTB4 and a novel LTB4 receptor antagonist radioligand [3H]CGS 23131 (LY223982). Saturation studies revealed that [3H]CGS 23131 labeled a single class of recognition sites with high affinity (Kd = 13 nM) and limited capacity (apparent Bmax = 2.8 pmol/10(7) cells). In contrast, [3H]LTB4 labeled both a set of high (Kd = 0.3 nM) and lower affinity (Kd = 5 nM) recognition sites. However, the apparent density of [3H]LTB4 binding to intact human PMN (combined Bmax = 380 fmol/10(7) cells) was approximately 14% of that observed with [3H]CGS 23131. In ligand competition studies, various LTB4 agonists and antagonists were found to inhibit the binding of [3H]CGS 23131, revealing a pharmacological profile consistent with the specific labeling of the LTB4 receptor. A positive rank order correlation (r = 0.79) was observed between the ligand competition profiles obtained with [3H]CGS 23131 and [3H]LTB4. Both LTB4 and its omega oxidation product, 20-OH-LTB4, were found to inhibit the binding of 1.0 nM [3H]CGS 23131 in a biphasic fashion consistent with the existence of multiple affinity components of the LTB4 receptor. In competing for 0.5 nM [3H]LTB4 binding, these compounds were found to produce monophasic inhibition curves, which was indicative of a selective interaction at the high-affinity LTB4 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Ibutilide, a new compound with potent class III antiarrhythmic activity, activates a slow inward Na+ current in guinea pig ventricular cells. Ibutilide, a class III antiarrhythmic compound under clinical development, was tested for specific activity on inward currents in guinea pig ventricular cells. Current clamp experiments showed a bell-shaped, dose-dependent effect of the compound on action potential duration and plateau height at concentrations beginning at 10(-9) M. In voltage clamp experiments in which the cells were bathed in K(+)-free solutions that contained external Na+0 and Ca++0, ibutilide, at 10(-8) M, increased a late inward current without affecting the fast inward Na+ current. The late inward current amplitudes, measured at 60 and 600 msec into a +20-m V step from -80 mV, were increased, respectively, from 0.73 +/- 0.07 to 1.1 +/- 0.06 nA, and from 0.02 +/- 0.02 to 0.15 +/- 0.03 nA, with a corresponding Kd of 2.3 x 10(-9) and 1.3 x 10(-9) M. However, if Na+0 was removed via appropriate ion substitution, the effect of ibutilide on the late inward current disappeared. On the other hand, when inward currents were maximized by the drug in Na+0 and Ca++0 containing external solution, removal of Na+0 reversibly removed a prominent late inward current which, when isolated from the L-type Ca++ current by current subtraction method, displayed a peak current potential of +30 mV and two decaying time constants of 38.2 +/- 3.8 and 200.8 +/- 43.3 msec at +20 mV. We concluded that ibutilide prolonged action potential duration through activation of a slow inward Na+ current. The identity of this current was discussed.


Increases in the oxytocin-induced prostaglandin F2 alpha response and reduction in the concentrations of endometrial oxytocin receptors in ewes in response to progesterone. Ovariectomized ewes were given progesterone and oestrogen priming as steroid pretreatment and subsequently treated with progesterone, prostaglandin F2 alpha (PGF2 alpha), or both. In Expt 1, plasma concentrations of the metabolite 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were measured after an i.v. injection of oxytocin. There was little PGFM response in the untreated control ewes or in the pretreated ewes. Treatment with PGF2 alpha alone had no effect (P greater than 0.05), whereas treatment with progesterone either alone or with PGF2 alpha significantly (P less than 0.05) increased the uterine PGFM response to oxytocin. In Expt 2, chronically ovariectomized ewes had high concentrations of endometrial oxytocin receptors. Treatment with PGF2 alpha alone did not alter the concentrations of the receptors. Treatment with progesterone either alone or with PGF2 alpha significantly (P less than 0.05) reduced the concentrations of the receptors. It is concluded that progesterone promotes the PGFM response to oxytocin, but simultaneously suppresses the concentrations of endometrial oxytocin receptors.


Independence of progesterone blockade of the luteinizing hormone surge in ewes from opioid activity at naloxone-sensitive receptors. Fifteen ovariectomized ewes were treated with implants (s.c.) creating circulating luteal progesterone concentrations of 1.6 +/- 0.1 ng ml-1 serum. Ten days later, progesterone implants were removed from five ewes which were then infused with saline for 64 h (0.154 mol NaCl l-1, 20 ml h-1, i.v.). Ewes with progesterone implants remaining were infused with saline (n = 5) or naloxone (0.5 mg kg-1 h-1, n = 5) in saline for 64 h. At 36 h of infusion, all ewes were injected with oestradiol (20 micrograms in 1 ml groundnut oil, i.m.). During the first 36 h of infusion, serum luteinizing hormone (LH) concentrations were similar in ewes infused with saline after progesterone withdrawal and ewes infused with naloxone, but with progesterone implants remaining (1.23 +/- 0.11 and 1.28 +/- 0.23 ng ml-1 serum, respectively, mean +/- SEM, P greater than 0.05). These values exceeded circulating LH concentrations during the first 36 h of saline infusion of ewes with progesterone implants remaining (0.59 +/- 0.09 ng ml-1 serum, P less than 0.05). The data suggested that progesterone suppression of tonic LH secretion, before oestradiol injection, was completely antagonized by naloxone. After oestradiol injection, circulating LH concentrations decreased for about 10 h in ewes of all groups. A surge in circulating LH concentrations peaked 24 h after oestradiol injection in ewes infused with saline after progesterone withdrawal (8.16 +/- 3.18 ng LH ml-1 serum).(ABSTRACT TRUNCATED AT 250 WORDS)


Energy metabolism of the human fallopian tube. The consumption of oxygen (QO2), the production of lactate and the profile of four key metabolic enzymes were measured in small samples of human oviductal mucosa (endosalpinx) removed at surgery. The QO2 in the absence of substrate was 3.4 microliters O2 (mg dry wt)-1 h-1, a value typical of quiescent tissue. The QO2 was stimulated by glucose, but diminished by glutamine and acetoacetate. Tissue lactate production was low and not increased by glucose. Hexokinase had the highest activity of the enzymes measured, followed by 2-oxoglutarate dehydrogenase; 6-phosphofructokinase and glycogen phosphorylase had low activities. The data are consistent with the proposition that glucose is a major metabolic fuel for human endosalpinx.


Microgeographic and temporal genetic variation in populations of the bluetongue virus vector Culicoides variipennis (Diptera: Ceratopogonidae). Seven Colorado populations of the bluetongue virus vector Culicoides varipennis (Coquillett) were analyzed for genetic variation at 19-21 isozyme loci. Permanent populations, which overwinter as larvae, showed little temporal genetic change at 19 loci. PGD and MDH showed seasonal changes in gene frequencies, attributable to selection at two permanent populations. Two temporary populations showed low heterozygosity compared with permanent populations. Independent estimates of gene flow, calculated using FST and the private allele method, were Nm* = 2.15 and 6.95, respectively. Colorado C. variipennis permanent populations showed high levels of gene flow which prevented significant genetic differentiation due to genetic drift. Temporary populations showed significant gene frequency differences from nearby permanent populations due to the "founder effect" associated with chance colonization.


Japanese encephalitis virus in Bangkok: factors influencing vector infections in three suburban communities. An unexpected outbreak of Japanese encephalitis (JE) in Bangkok in 1985 led us to investigate the vector ecology of urban JE from January 1986 to June 1987 at three suburban sites that displayed a wide range of factors imputed to influence JE transmission. Culex tritaeniorhynchus Giles and Cx. gelidus Theobald, suspected vectors, comprised 71-96% of all mosquitoes collected by CO2-baited CDC traps at the three sites. Mean of mosquito abundance per two trap-nights per month ranged from 28 to 5,728 mosquitoes at the sites of lowest and highest abundance, respectively. Cx. tritaeniorhynchus yielded more JE isolates (n = 16) than Cx. gelidus (n = 7), but the minimum infection rates of the two species (number of JE isolates per 1,000 mosquitoes tested; MIR, 0.17 and 0.47, respectively) were comparable and covaried with vector abundance. Moreover, the proportion of sentinel pigs that had JE antibodies generally increased proportionately with vector abundance at the sites. Vector abundance was high in monsoon (May-October), moderate in transition (March-April and November-December), and low in dry (January-February) seasons. Mosquitoes collected in monsoon seasons yielded 96% of the JE isolates, whereas 4 and 0% of the isolates were obtained from transition and dry season collections, respectively. More pigs seroconverted in monsoon and transition seasons than in dry seasons. Indices of JE transmission activity (vector abundance, pig seroconversions, and MIRs) increased proportionately with rainfall. Despite higher indices at the site of greatest vector abundance than elsewhere, the risk of human infection appeared greatest at the site with moderate vector abundance because of its greatest human population density.


Biphasic increase of apical Cl- conductance by muscarinic stimulation of HT-29cl.19A human colon carcinoma cell line: evidence for activation of different Cl- conductances by carbachol and forskolin. The modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10(-4) M carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15 +/- 1 mV from a resting value of -52 +/- 1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22 +/- 1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11 +/- 1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6 +/- 1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4 +/- 0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl- conductance in the apical and basolateral membrane, the second phase by increased K+ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl- conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl- channels are involved in the carbachol response: one Ca2+ dependent and a second which may be PKC sensitive. In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl- conductance. It is concluded that the short-lasting carbachol/Ca(2+)-dependent Cl- conductance is different from the forskolin-activated conductance. The increase of the Cl- conductance in the presence of forskolin by carbachol may be due to activation of different Cl- channels or to modulation of the PKA-activated Cl- channels by activated PKC.


Amiloride in ouabain-induced acidification, inotropy and arrhythmia: 23Na & 31P NMR in perfused hearts. The increase in intracellular sodium (Nai), resulting from inhibition of the Na/K ATPase by cardiac glycosides, is known to increase calcium influx via Na(+)-Ca2+ exchange, and thereby increase contractility. This increase in intracellular Ca2+ has been related to the development of intracellular acidification and enhanced activity of the Na(+)-H+ exchanger as a measure by the cell to prevent further acidification. Thus, the efflux of the H+ ions results in an additional increase in Nai. This may subsequently lead to an increased rate of Ca2+ influx and therefore to the potentiation of the effects of cardiac glycosides. To assess the role of Na(+)-H+ exchange in the mechanism of ouabain action in the beating heart we used amiloride, a known inhibitor of Na(+)-H+ exchange. Isolated rat hearts were perfused with either ouabain (50 microM) alone (n = 8, Group I), amiloride (1.0 mM) + ouabain (50 microM) (n = 8, Group II), or amiloride (1.0 mM) alone as a control group (n = 4, Group III). 23Na and 31P NMR spectroscopy were used to assess the changes in Nai and intracellular pH (pHi), respectively, while simultaneous and continuous monitoring of left ventricular pressure was carried out. Perfusion with both ouabain alone (Group I) or ouabain + amiloride (Group II), resulted in a time dependent increase in Nai levels, reaching (within 25 mins) a maximum of 200 +/- 7% of control in Group I, and 170 +/- 10% of control in Group II. Concurrently, a mild but significant decrease in pHi was observed in both groups. This decrease, however, was significantly higher in Group II compared to Group I (0.34 pH units vs. 0.19 pH units, respectively; P less than 0.05), suggesting that inhibition of Na(+)-H+ exchange by amiloride limits the recovery from ouabain-induced intracellular acidification. While developed pressure gradually increased in Group I to a maximum of 268 +/- 52% of control, the addition of amiloride in Group II substantially reduced the positive inotropic effect. Ventricular fibrillation (VF) developed in three of the eight hearts in Group I within 10-13 mins after the addition of ouabain. Interestingly, the rate of Nai increase in hearts that sustained VF was significantly higher compared to those without VF (mean slope 10.1 +/- 2.11 vs. 3.9 +/- 1.0, respectively; P less than 0.0001). Ventricular fibrillation did not develop in Group II or III.(ABSTRACT TRUNCATED AT 400 WORDS)


Myocardial Na+,K(+)-ATPase in tachycardia induced cardiomyopathy. Na+,K(+)-ATPase is a major determinant of myocyte homeostasis and excitation-contraction. Cardiac glycosides such as digitalis and ouabain increase the inotropic state of the heart through the inhibition of Na+,K(+)-ATPase. While cardiac glycosides are commonly used in the setting of congestive heart failure, optimal therapy would depend upon an intact Na+,K(+)-ATPase system. Changes in Na+,K(+)-ATPase activity and glycoside receptor density with the development of cardiomyopathy have not been well defined. Accordingly, left ventricular (LV) function and Na+,K(+)-ATPase activity and glycoside binding were examined in 7 pigs with dilated cardiomyopathy and in 7 controls. Dilated cardiomyopathy was produced by pacing induced supraventricular tachycardia (SVT) for 3 weeks at 240 bpm. Left ventricular function was examined by simultaneous echocardiography and catheterization. Left ventricular fractional shortening significantly decreased with SVT (34 +/- 2 vs. 10 +/- 2%, P less than 0.05) and LV diastolic dimension and pressure significantly increased (3.8 +/- 0.3 vs. 5.1 +/- 0.4 cm, and 8 +/- 2 vs. 27 +/- 2 mmHg, respectively, P less than 0.05) as compared to controls. Na+,K(+)-ATPase activity was assayed as potassium dependent p-nitrophenol-phosphatase activity. Glycoside receptor density (Bmax) and affinity (KD) was determined using [3H]-ouabain binding assays. Na+,K(+)-ATPase activity, Bmax, and KD all significantly fell from control values with SVT induced cardiomyopathy (0.64 +/- 0.06 vs. 0.45 +/- 0.12 micrograms pNP/mg/h, 5.5 +/- 0.4 vs. 1.9 +/- 0.4 pmol/mg, and 15 +/- 3 vs. 9 +/- 3 nM, respectively, P less than 0.05). The distribution of Na+,K(+)-ATPase in LV sections taken from control and SVT hearts were examined using immunohistochemical techniques. A patchy distribution of Na+,K(+)-ATPase along the sarcolemma in SVT sections was observed as opposed to a more uniform distribution in control myocytes. There was no observable change in the relative content and distribution of the Na+,K(+)-ATPase isoforms alpha 2 and alpha 3 in the SVT sections as compared to controls. In an additional set of experiments, changes in LV as well as isolated myocyte responsiveness to ouabain were examined. Left ventricular fractional shortening and peak dP/dt were measured following administration of 20-60 micrograms/Kg of ouabain in control (n = 3) and SVT (n = 3) pigs. In the control group, 40 micrograms/Kg caused a 25% in LV fractional shortening and a 60% increase in peak dP/dt from baseline. Cumulative doses of 60 micrograms/Kg in the control pigs resulted in over a 75% increase in peak dP/dt from baseline values.(ABSTRACT TRUNCATED AT 400 WORDS)


Neural, humoral, and metabolic control of coronary vascular resistance during exercise. Neural, humoral, and metabolic effects on coronary vascular resistance were examined during exercise in conscious dogs, chronically instrumented for the measurement of aortic pressure, heart rate, and left circumflex coronary blood flow. Exercise significantly decreased coronary vascular resistance (CVR) in intact (INT) group, in which CVR was controlled by neural, humoral, and metabolic factors. In cardiac denervated (CD) group with pretreatment of alpha blocker (phentolamine, 2 mg/kg), in which CVR was controlled only by metabolic factor, exercise significantly decreased CVR. To eliminate metabolic effect on CVR, CVR was normalized by the product of CVR and double product (DP = mean aortic pressure x heart rate). CVR.DP did not change throughout the exercise in dogs with CD with alpha blocker. Thus, metabolic effect on CVR during exercise can be excluded by the product of CVR and DP. This calculation was applied to INT group with alpha blocker and CD group without blockers. The 12 km/h exercise significantly increased CVR.DP from 4.1 +/- 0.3 x 10(4) to 6.4 +/- 0.9 x 10(4) in INT group with alpha blocker, in which CVR was controlled only by neural factor, and from 2.8 +/- 0.2 x 10(4) to 4.5 +/- 0.5 x 10(4) in CD group, in which CVR was controlled only by humoral factor. These data suggest that 1) neural and humoral coronary vasoconstriction occurs during exercise, 2) neural and humoral vasoconstriction is overwhelmed by metabolic vasodilation, and 3) CVR.DP is a beneficial calculation for excluding metabolic effect on CVR during exercise.


Increased responses to adenosine and 2-chloroadenosine of cyclic AMP-generating systems in the primary cortical region of cobalt-induced epilepsy in the rat. The epileptic activity was induced by an injection of cobalt chloride solution into the unilateral sensorimotor cortex of rats. Cyclic AMP accumulations elicited by adenosine and 2-chloroadenosine were investigated in slices from different regions of the epileptic cortex. The cyclic AMP accumulation was increased in the primary cortical region of cobalt-induced epilepsy. The greatest increase in cyclic AMP accumulation was detected between 2 and 3 weeks after treatment by cobalt, in conjunction with the electrographic and behavioral findings. The results suggest the functional significance of cortical cyclic AMP-generating systems in cobalt-induced epilepsy.


New fluorescent probes E3810 and methoxy E3810 for determining distributions of the apical membrane and the acidic compartment of gastric acid secreting cells. Substituted benzimidazoles such as omeprazole, E3810 and methoxy E3810 were inhibitors of gastric H+, K(+)-ATPase which is rich in the apical membrane of gastric parietal or oxyntic cells at the secreting state. The acid-activated compounds of omeprazole and methoxy E3810, which have methoxy group at the 5-position in the benzimidazole ring, are fluorescent (excitation wavelength = 370 nm; emission wavelength = 560 nm). The fluorescence disappeared when the activated compounds reacted with the ATPase or glutathione. Using this fluorescence property, the distribution of the intracellular acidic canalicular space in isolated single parietal cells was determined. On the other hand, irradiation with ultraviolet light (335 nm) of the acid-activated compound of E3810 which had been reacted with sulfhydryl group of the ATPase or glutathione resulted in a formation of a fluorescent compound (emission = 470 nm). Using this second fluorescence property, we determined the distribution of the apical membrane of the intracellular canaliculus of isolated single mammalian parietal cells and also the location of the apical membrane on the external surface of newt oxyntic cells.


[A case report of multiple pulmonary tumors as a sole manifestation of synovial sarcoma]. A 47-year-old woman was admitted to our hospital for cough and dyspnea. Roentgenologic studies and bronchoscopy revealed multiple lung tumors one of which obstructed the right main bronchus. Right pneumonectomy was performed for the pending obstruction of the trachea. The tumor in the right S1 was found to be protruding into the trachea through the right B1 and the main bronchus in a polypoid fashion. The pathological diagnosis of synovial sarcoma was made on the basis of the characteristic biphasic structure composed of spindle cells and epithelioid cells forming gland-like spaces. Three years and eight months after the pneumonectomy, a nodule in the tendon of the extensor hallucis longus muscle became palpable. It was also a synovial sarcoma pathologically. Synovial sarcoma is a soft tissue sarcoma which usually arises in the extremities. It is very rare for pulmonary metastasis of this tumor to be found while the primary tumor is undetectable.


[Type III procollagen N-terminal peptide (P-III-P, prolyl hydroxylase (PH), and laminin P1 levels in serum and BALF of radiotherapy patients]. The etiology of pulmonary fibrosis remains unclear, and at present there are no definite biochemical markers of its activity. We measured serum and BALF levels of type III procollagen N-terminal peptide (P-III-P), prolyl hydroxylase (PH), and laminin P1 in patients who had undergone radiotherapy for malignant neoplasms, and investigated their value as biochemical markers in a model of pulmonary fibrosis. The following results were obtained: 1) Patients with abnormal liver function had significantly higher serum P-III-P levels and showed a tendency to have higher serum PH levels. If P-III-P or PH are to be used as markers of pulmonary fibrosis, the effect of liver function must be taken into consideration; however, no significant difference was detected with respect to laminin P1 levels. 2) Serum P-III-P levels were significantly elevated by radiotherapy. 3) Laminin P1 levels rose in a similar manner to P-III-P levels after radiotherapy, but no significant change was detected. 4) In most cases, the levels of all markers in BALF were below the threshold of detection, nevertheless all three markers were elevated in a patient who developed diffuse radiation pneumonitis during radiotherapy. Increases in the lymphocyte count were found in BALF of this patient. 5) BALF hyaluronic acid levels were negative in the 3 cases assayed. 6) A significant correlation between P-III-P and laminin P1 in serum was shown, but no significant correlations could be found between the other combinations of markers in serum. Thus it appears that serum P-III-P and laminin P1 are valid biochemical markers of pulmonary fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)


[Imprint cytodiagnosis of lymph node metastasis in resected lung cancer]. The cytological examination of lymph node imprints is a rapid and useful method for diagnosis of lymph node metastasis. We applied this technique for the diagnosis of regional lymph node metastasis of resected lung cancer, and compared its diagnostic value with histological examination. The accuracy rate of imprint cytology is 97.3%, and the sensitivity rate is 100%. We conclude that imprint cytology of lymph node for diagnosis of cancer metastasis is highly accurate and simple, and its diagnostic value may be equal to that of routine histological examination.


Autoantibodies to myeloperoxidase in brown Norway rats treated with mercuric chloride. Mercuric chloride (HgCl2) induces an autoimmune syndrome in Brown Norway (BN) rats characterized by the presence of a number of autoantibodies, including antibodies to glomerular basement membrane. Tissue injury has previously been reported to be rare in this model, but in the accompanying paper we describe changes in a number of organs including a necrotizing leucocytoclastic vasculitis in the gut. Myeloperoxidase (MPO) is one of the target antigens for anti-neutrophil cytoplasm antibodies, that are present in the majority of patients with the human autoimmune disease systemic vasculitis and have been implicated in pathogenesis. There is at present, no animal model for anti-neutrophil cytoplasm antibody positive systemic vasculitis. Ten BN rats were given five injections of HgCl2, each of 1 mg/kg, over 10 days. Sequential serum samples were tested for autoantibodies to MPO using solid phase assays, indirect immunofluorescence on normal rat neutrophils, and Western blot analysis. The specificity of these antibodies in the solid phase assay was confirmed by inhibition studies with purified antigen, and by testing binding to uncoated plates. Sera from control animals treated with saline were also tested. BN rats given HgCl2 developed antibodies to MPO, which in Western blots bound to similar determinants to those bound by human anti-MPO antibodies. The anti-MPO antibodies resolved spontaneously, with a time course similar to that of the anti-glomerular basement membrane antibodies, but there was no correlation between the two antibody responses in individual animals. There was no anti-MPO activity in sera taken before HgCl2 was given, nor in sera from saline-treated controls. BN rats treated with HgCl2 develop anti-MPO antibodies. Together with the description in the accompanying paper of necrotizing vasculitis in these animals, these observations suggest that HgCl2-induced autoimmunity in the BN rat may provide a useful model of anti-neutrophil cytoplasm antibody positive systemic vasculitis.


Epstein-Barr virus-associated lymphoproliferative disorders. Herein we have provided a panorama of the clinical, histopathologic, and molecular biologic mechanisms of EBV-induced LPD particularly in immunosuppressed individuals. A listing of EBV-related diseases is shown in Table 4. We have stressed the frequent need to use multiple diagnostic methods for detecting EBV genome, particularly in immunodeficient patients who may fail to mount antibody responses to EBV. Given that we now recognize some of the immunocompromised patient populations at high risk for EBV-induced LPD, and have developed techniques for detecting EBV genome and early LPD, we may eventually prevent the occurrence of some of these life-threatening diseases. For example, we have learned to recognize and distinguish hepatic allograft rejection from EBV-induced LPD in hepatic biopsies (154). A periportal and sinusoidal infiltrate of small and large lymphoid cells, immunoblasts, and plasma cells, alert us to stain frozen liver sections for EBNA. Finding EBV guides the clinicians to reducing immunosuppression which then allows the restoration of immunosurveillance against the EBV-infected B cells. Whether an EBV vaccine can be successful in immunosuppressed individuals remains to be seen. As for other vaccines, many logistical problems prevail, such as the early occurrence of EBV infection during infancy in regions where BL is endemic. Surely, with the menacing threat that approximately 10% of patients with AIDS will develop NHL, new anti-viral therapy against EBV and the causative agent of AIDS and HIV, will be developed. The pathologist and virologist play essential roles in the recognition of EBV infection by performing clinical laboratory determinations. The characteristic histopathologic features of EBV-induced LPD are now recognized and when confirmed with molecular hybridization and immunofluorescent techniques will provide a solid diagnostic approach and, thus, a foundation for developing a sound therapeutic strategy.


Increased cytotoxicity of polyunsaturated fatty acids on human tumoral B and T-cell lines compared with normal lymphocytes. Epidemiological and experimental data suggest that fatty acids may modulate the growth of tumor cells. We have analyzed the effect of different types of fatty acids, bound to serum proteins in physiological conditions, on the lipid composition and growth of human neoplastic B and T-cell lines and compared their effect on normal lymphocyte proliferation. Fatty acids with 0 to 2 unsaturations (stearic, oleic, and linoleic), at concentrations up to 50 or 100 microM did not significantly affect the proliferation of leukemic cells. However, long-chain polyunsaturated fatty acids (PUFA), and mainly docosahexaenoic (22:6, n-3), were cytotoxic at concentrations greater than or equal to 20 microM after 48-72 h in culture. Simultaneous supplementation with vitamin E restored normal cell growth. The amount of end-products of lipid peroxidation in cells correlated with the observed toxicity but the amount of superoxides did not. Fatty acid supplementations increased cell triacylglycerol content but did not affect the degree of unsaturation of phospholipids, cholesterol/phospholipids molar ratio, or membrane fluidity. Glutathione-S-transferase activity was low in Raji and CEM cells, moderate in lymphocytes and high in Ramos cells and did not increase with supplementations. The proliferation of normal lymphocytes, which produced lower amounts of end-products of lipid perodixation, was not inhibited, but in some cases stimulated, by PUFA (with the exception of 30 microM 22:6). The extension of these results to situations in vivo could lead to use of PUFA for delaying leukemia progression or in adjuvant chemotherapy.


ACTH accelerates the attenuation of alpha 2-adrenoceptors response in nucleus accumbens following chronic desipramine. The inclusion of clonidine (CLO) induced a dose-dependent reduction of K(+)-evoked [3H] dopamine ([3H]DA) release in slices from rat nucleus accumbens. This inhibition was clearly attenuated in animals previously administered desipramine daily (DMI, 10 mg/kg i.p.) during 21 days, but not in rats submitted to a persistent treatment with DMI during 10 days. However, the coadministration of adrenocorticotrophic hormone (ACTH, 50 IU/kg s.c.) and DMI (10 mg/kg i.p.) for 10 days provoked a clear decrease in the inhibition produced by alpha 2-adrenoceptor stimulation, while ACTH alone had no effect. These results may indicate that ACTH accelerates the onset of DMI-induced adaptive changes on central alpha 2-adrenoceptor in the mesolimbic area.


Intracerebroventricular 5,7-DHT alters the in vitro function of rat cortical GABAA/benzodiazepine chloride ionophore receptor complexes. We have earlier presented data indicating that the anxiolytic-like effect obtained in rats after depletion of brain 5-HT by means of PCPA or 5,7-DHT treatment is indirect and appears to involve the GABAA/benzodiazepine chloride ionophore receptor complex (GABAA/BDZ-RC), and that it is abolished by adrenalectomy. In the present series of experiments we have therefore investigated the 36Cl(-)-uptake in rat synaptoneurosomal preparations of central cortices from 5,7-DHT- and SHAM-lesioned animals. The GABA as well as the 3 alpha,5 alpha-tetrahydrodeoxycorticosterone (THDOC) induced picrotoxin-sensitive increase in 36Cl(-)-uptake was significantly lower than that observed in the SHAM-lesioned animals, indicating that the 5,7-DHT lesion has rendered the GABAA/BDZ-RC subsensitive to two of its tentative endogenous ligands. This effect of the 5,7-DHT lesion on the function on the GABAA/BDZ-RC was reversed by adrenalectomy, indicating that an intact adrenocortical function is required for the development of GABAA/BDZ-RC subsensitivity in 5,7-DHT-lesioned rats. A tentative conclusion of these findings is that the 5,7-DHT lesion induces an increase in release of GABA and/or barbiturate-like steroids and that this increase is reversed by adrenalectomy. The findings from these in vitro studies parallel those from our previous behavioral experiments and provide further support for the notion that a decreased serotonergic influence in the central nervous system may, possibly via the adrenocortical system, enhance the function of the GABAA/BDZ-RC.


Increased glomerular atrial natriuretic peptide receptor affinity in experimental nephrotic syndrome. Atrial natriuretic peptide (ANP) is a cardiac hormone with natriuretic and diuretic effects. To better define the ANP hormonal system in the nephrotic syndrome, a condition associated with renal sodium retention, we undertook a study of glomerular ANP receptors in rats with puromycin aminonucleoside-induced nephrotic syndrome and in pair-fed controls. Nephrotic rats had significantly decreased serum albumin and total protein and significantly increased serum cholesterol, triglycerides and 24 hour urinary protein excretion. Plasma level of atrial natriuretic peptide was similar in both groups of rats. Competition binding inhibition studies in isolated glomeruli demonstrated one binding site in both groups of rats. The density of ANP binding sites in isolated glomeruli was similar in nephrotic and pair-fed rats while the binding affinity was increased significantly in the nephrotic rats. This is the first study to demonstrate alterations in renal ANP receptors in the nephrotic syndrome. Further studies will be necessary to determine whether alterations in glomerular ANP receptors contribute to renal sodium retention in the nephrotic syndrome.


Localization of mRNA for protein phosphatase 2C in the brain of adult rats. Type 2C protein phosphatase (PP2C) is one of four major serine-threonine specific phosphoprotein phosphatases which modulate various intracellular activities. By in situ hybridization analysis of the adult rat, expression signals of mRNA for PP2C were observed most highly in the granule cells and Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and granule cells of the dentate gyrus, and plexus choroideus of the lateral ventricle, whereas moderate levels of its expression were observed in the medial habenula, piriform cortex and the pineal body. Several discrete nuclei of the brainstem including pars compacta of the substantia nigra, the pontine nuclei, and the locus ceruleus expressed the mRNA moderately. Weak expression of PP2C mRNA was observed in mitral and internal granule cells of the olfactory bulb, spinal cord gray matter, the cerebral neocortex, thalamic and hypothalamic nuclei. Only faint expression was detected in the caudate putamen. These patterns of expression are different from that of calcineurin/PP2B reported by other immunohistochemical studies and it is suggested that various neuronal proteins are differentially dephosphorylated by the different types of PP.


Effects of insulin and insulin-like growth factors on neurofilament mRNA and tubulin mRNA content in human neuroblastoma SH-SY5Y cells. Insulin-like growth factors (IGFs) are implicated in the development of the vertebrate neural circuitry, and increase neurite growth in vitro and in vivo. The construction of the cytoskeleton is necessary for growth of axons and dendrites, and the neurofilament (NF) 68 kDa and 170 kDa proteins assemble to help form major fibrillar elements of the neurite cytoskeleton. We report that physiological concentrations of insulin, IGF-I or IGF-II increased the contents of 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs, relative to total RNA, in cultured human neuroblastoma SH-SY5Y cells. In contrast, the relative contents of histone 3.3 mRNA, and poly(A)+ RNA were not increased. Ligand concentrations which increased NF mRNAs were very similar to those which increased neurite outgrowth. Although each gene was evidently independently regulated, the 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs were nevertheless all transiently elevated over approximately the same time interval in response to insulin. These data, when considered together with studies by others with nerve growth factor, show that the 68 kDa and 170 kDa NF mRNAs are elevated in a biochemical pathway activated in common during neurite outgrowth directed by insulin, IGF-I, IGF-II, and nerve growth factor.


Expression of c-fos and NGFI-A messenger RNA in the medulla oblongata of the anaesthetized rat following stimulation of vagal and cardiovascular afferents. Messenger RNA encoding the immediate early genes (IEGs) c-fos and NGFI-A was localized by in situ hybridization of specific 35S-labelled oligonucleotides to detect activated neurones in the medulla oblongata following unilateral electrical stimulation of the vagus (nX) and aortic depressor nerve (ADN), and following mechanical stimulation of the left carotid sinus (CS). In electrically stimulated rats, c-fos and NGFI-A mRNA was strongly expressed in the nucleus tractus solitarius (NTS) (predominantly ipsilaterally), area postrema (AP) and in a dorsal subregion of the paratrigeminal nucleus (PTN). Lower levels of c-fos and NGFI-A mRNA were seen in the ipsilateral NTS and PTN following mechanical stimulation of the left CS. In general these data correlate with the topography of innervation by the different nerve afferents, although the expression in the PTN (and in some cases the AP) would not be predicted on the basis of neuronal innervation patterns reported for the rat. Expression of these IEGs also occurred in the rostral and caudal ventrolateral medulla and inferior olive of both stimulated and sham-operated rats; presumably due to effects of the anaesthesia and surgical procedures. In conclusion the localization of the expression of c-fos and NGFI-A mRNAs represents a useful neuroanatomical technique for detecting the cell bodies of neurones that are activated by cardiovascular nerve afferents and should allow the further characterization of the neurochemical identity of these neurones.


Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase. Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the conversion of norepinephrine to epinephrine, the last step of catecholamine biosynthesis. We have previously reported molecular cloning of cDNA encoding human PNMT and chromosomal localization of its gene (Kaneda et al., J. Biol. Chem., 263 (1988) 7672-7677). In this report, we isolated the chromosomal gene encoding mouse PNMT by cross-hybridization with the human PNMT cDNA. Mouse PNMT gene spanned about 1.8 kb and consisted of 3 exons. Primer extension analysis showed two putative transcription initiation sites. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of the mouse PNMT mRNA in brain (pons and medulla oblongata) and adrenal gland. Subsequently cDNA encoding mouse PNMT was amplified by RT-PCR and cloned into the plasmid vector. Mouse PMNT gene contained the protein-coding region of 885 bp (295 amino acids) with the predicted molecular weight of 32,627. The deduced amino acid sequence of mouse PNMT revealed the major difference in the N-terminal region, as compared to the human and bovine PNMT sequences. In the 5'-terminal region of the mouse PNMT gene, we found the existence of 23 bp direct repeat sequences, which was not observed in the corresponding regions of the human and bovine PNMT genes. The presence or absence of the direct repeats caused the major difference in the PNMT sequences among species. The typical TATA, GC, and CACCC boxes as well as several sequences homologous to glucocorticoids response elements (GRE) were located in the 5'-flanking region of the mouse PNMT gene.


Identification of an alternatively spliced avian member of the synaptophysin gene family. Synaptic vesicle membrane proteins are important in the release of neurotransmitters and as markers of presynaptic differentiation in neurons, and the synaptophysins are a major class of synaptic vesicle proteins. By low stringency screening of a chick brain cDNA library with a rat synaptophysin probe, we have isolated cDNAs that encode a novel member of the synaptophysin/synaptoporin family. Two different protein-coding forms of the cDNA were found, apparently generated through alternative splicing of a single gene. The deduced proteins, called synaptophysin IIa and synaptophysin IIb, share 258 amino acids (starting from position 10 in IIa and position 30 in IIb), that are most closely related to the rat synaptoporin sequence. The N-terminal sequence of IIa is similar to that of rat synaptoporin, and the N-terminal sequence of IIb is similar to that of rat synaptophysin. Northern blot analysis and nuclease protection experiments demonstrate that IIa and IIb are expressed in a variety of brain regions, the spinal cord, and dorsal root ganglia, but not in non-neuronal tissues. Further, the two splice variants are differentially distributed. In most brain regions the IIb form predominates, and the cerebellum appears to express only the IIb form, but the IIa form is relatively elevated in peripheral neurons. Western blot analysis with an antibody to a synthetic peptide common to both forms demonstrates the expression of synaptophysin II as a 39 kDa protein, apparently distinct from synaptophysin (40 kDa). The results suggest that the regulation and function of the synaptophysin gene family is more complex than had been appreciated.


Sequences on the N-terminus of ACh receptor subunits regulate their assembly. Mutant alpha subunits of Torpedo acetylcholine receptors (AChR) were constructed and expressed in Xenopus oocytes together with other normal subunits to investigate regions in the subunit that are required for subunit assembly. I have found that chimeric alpha subunits, consisting of the N-terminal extracellular domain of the AChR alpha subunit, followed either by the hydrophobic transmembrane segments of GABAA receptor or glutamate receptor subunits, were still recognized as the AChR subunit and associated with co-expressed other normal AChR subunits, suggesting that this part of the N-terminal extracellular domain contains 'assembly signals'.


Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus. The basal expression of the protein products of the inducible immediate early genes (IEGs), Fos, Jun, and Krox 24, was investigated in rat hippocampus using immunocytochemical visualization methods with antisera specific for Fos only, Fos and the Fos-related antigens (FRAs), the Jun family, and Krox 24 (previously described as TIS 8, egr-1, NGF-IA or zif 268). In the normal adult rat brain basal levels of Jun, Krox 24 and Fos-related antigens but not Fos were seen within the hippocampus. More specifically very high basal levels of Jun were seen in the dentate granule cells with high basal Krox 24 levels seen in the CA1-subiculum region of the rat hippocampus. Basal FRAs but not Fos-positive cells were seen at low levels in the dentate granule cells. The implications of these results to the functioning of IEG proteins in hippocampal neurons is discussed.


Phosphodiesters in saponified extracts of human breast and colon tumors using 31P magnetic resonance spectroscopy. Saponified phospholipid extracts of malignant and normal human breast and colon surgical tissue specimens (n = 45) generate characteristic phosphodiester profiles using 31P magnetic resonance (MR) spectroscopy. The resultant 31P MR spectroscopic profiles of the analyzed tissues are used to differentiate malignant from normal. The appearance of an uncharacterized resonance at 0.29 delta in the malignant tissue spectra (50% of breast and 75% of colon specimens) is the most notable qualitative finding. Quantitatively, malignant colon tissues differ from normal colon tissues with depressed levels of phosphatidylserine and elevated levels of glycerol 3-phosphorylglycerol and an index measuring the summation of phospholipid polar head group residues with free hydroxyl groups. Malignant breast tissues have significantly elevated levels of glycerol 3-phosphorylethanolamine and significantly depressed levels of glycerol 3-phosphorylcholine compared to normal breast tissues, reflecting a perturbation in the balance of lipid residues that are the respective breakdown products of phosphatidylethanolamine and phosphatidylcholine. The concentration of the polar head group residues is compared to 31P MR spectroscopic profiles of colon and breast tissue phospholipids, in order to demonstrate the quantitative nature of the technique employed.


Starvation-inducible loci of Salmonella typhimurium: regulation and roles in starvation-survival. Four starvation-inducible loci (stiA, stiB, stiC, and stiE) of Salmonella typhimurium have been extensively characterized as to their genetic and physiologic regulation, and their roles in survival during prolonged simultaneous phosphate (P)-, carbon (C)- and nitrogen (N)-starvation (PCN-starvation). Strains of S. typhimurium LT-2, isogenic with the exception of lacking either the stiA, stiB or stiC locus, died off more quickly and survived at much reduced levels compared with their wild-type parent. When certain sti mutations were combined in the same strain, we found that viability of these cultures declined even more rapidly, and starvation-survival was affected to levels over-and-above the additive effects of each individual mutation, indicating an epistatic relationship between these loci. All four sti loci were, directly or indirectly, under negative control by the crp gene product (cAMP receptor protein, CRP). With the exception of stiB, all were similarly regulated by the cya gene product (i.e., cAMP). This suggests that CRP acts alone, or with a signal molecule other than cAMP, to cause repression of the stiB locus. In addition, all four loci are under positive regulation by the relA gene product (i.e., ppGpp) during C- or N-starvation, but not P-starvation. Since not all relA-dependent sti loci are induced during both C- and N-starvation, we propose that two separate ppGpp-dependent pathways function during C-starvation and N-starvation, respectively. Possible models for separate P-, C- and N-starvation-induction pathways are discussed.


Transcriptional regulation of the plastocyanin and cytochrome c553 genes from the cyanobacterium Anabaena species PCC 7937. The effect of copper on the levels of plastocyanin (PC) and cytochrome c553 (cyt-c)-specific transcripts from Anabaena sp. PCC 7937 was investigated. The addition of copper resulted in a marked increase in PC mRNA levels, and a decrease in cyt c mRNA levels. Thus the functional exchange between PC and cyt c seems to be regulated at the mRNA level. The copper-dependent increase in PC and decrease in cyt c mRNA levels was abolished when chloramphenicol was added to the cells. This suggests that de novo synthesis of at least one trans-acting element is required to regulate PC and cyt c mRNA levels. Both PC and cyt c mRNA stability was found to be unaltered under varying Cu2+ regimes. This leads to the conclusion that expression of both genes is regulated at the level of initiation of transcription.


Identification and characterization of a novel insertion sequence, IS1106, downstream of the porA gene in B15 Neisseria meningitidis. Examination of Neisseria meningitidis strains associated with endemic meningococcal disease demonstrated differences in the number of copies of a repetitive sequence. Characterization of a copy of this repetitive sequence present in B15 strains has revealed the presence of a novel insertion sequence (IS1106) located within a complex repetitive region downstream of the gene for the major surface antigen (porA). IS1106 has a length of 1137 bp and is flanked by 36bp inverted repeats. Two open reading frames (ORF1 and ORF2) are present in opposite strands in codon-codon register with ORF2 entirely located within ORF1. The predicted protein from ORF1 demonstrates homology with the 5A protein of IS5 (Kroger and Hobom, 1982). Strains from two independent outbreaks of B15 meningococcal disease in the UK were found to contain the same genomic deletion removing a copy of IS1106 downstream of the porA gene.


The cost of treating small cell lung cancer. To determine the cost of treating small cell lung cancer (SCLC) and to assess quality-adjusted survival in these patients. Retrospective analysis. Westmead Hospital, a tertiary referral institution. Consecutive sample of 31 patients with histologically proved SCLC, treated between January 1987 and December 1987. The cost of investigation, hospitalisation, chemotherapy, radiotherapy and follow-up of patients overall and for those with limited and extensive disease respectively. Quality-adjusted survival was based on a Q-TWiST analysis. The median overall cost per patient was $14,413 (range, $1188-$39,598) for all patients and for limited disease and extensive disease was $18,234 (range, $1914-$39,598) and $13,177 (range, $1188-$32,798) respectively. The two major costs were hospitalisation (42%) and chemotherapy (18%). Radiotherapy accounted for 11% of all costs. The Q-TWiST analysis suggests that for patients with limited disease, quality-adjusted survival is similar to absolute survival. The treatment of SCLC at our institution was expensive but the cost may be reduced by reduction in the duration of hospitalisation, the use of less expensive combination drug regimens, or the use of "true" outpatient chemotherapy. Despite intensive therapy, patients with limited disease maintained a reasonable quality of life.


Myasthenia gravis and pipecuronium--report of two cases. The use of pipecuronium in two patients with myasthenia gravis undergoing thymectomy is described. Neuromuscular function was monitored throughout using the train-of-four (TOF) mechanical twitch response. The cumulative dose-response to pipecuronium was determined in both patients during nitrous oxide-oxygen-narcotic anaesthesia. Both patients were sensitive to pipecuronium. The ED50 doses of pipecuronium were 11.6 and 11.1 micrograms.kg-1 and the ED95 doses were 35 and 33.3 micrograms.kg-1 in patients #1 and 2 respectively. Edrophonium 1 mg.kg-1 and neostigmine 0.06 mg.kg-1 were administered to patients #1 and 2 respectively for antagonism of residual neuromuscular blockade at 25 per cent spontaneous recovery of first twitch (T1) of the TOF stimulation. As with other non-depolarizing muscle relaxants pipecuronium in reduced dosage and with careful neuromuscular monitoring can be used to provide surgical relaxation safely in patients with controlled myasthenia gravis.


Increased platelet sodium-proton exchange rates in insulin-dependent (type 1) diabetic patients with nephropathy and hypertension. In order to assess the potential role of the plasma membrane sodium-proton (Na+/H+) exchanger in the pathogenesis of diabetic nephropathy, we investigated 32 insulin dependent (type 1) diabetic patients and 21 control subjects. We tested the Na+/H+ exchange as the rate of amiloride sensitive and sodium dependent volume gain of platelets suspended in sodium propionate. Patients with diabetic nephropathy had significantly increased rates of Na+/H+ exchange (0.31 +/- 0.06 s-1 x 10(-2)) when compared to those without nephropathy (0.24 +/- 0.07, p less than 0.05) or to a control group (0.23 +/- 05, p less than 0.05). Nine patients who were classified as hypertensive had a highly significant increase in the Na+/H+ exchange rates when compared to 23 non-hypertensive diabetic patients: 0.33 +/- 0.04 versus 0.24 +/- 0.06 (p less than 0.001). There was no significant correlation between the Na+/H+ exchange rates and age, diabetes duration, glycated hemoglobin or fructosamine levels on the day of the test. In summary, the data presented here demonstrate an increase in the Na+/H+ exchange rate in insulin-dependent diabetic patients with nephropathy and hypertension.


Alterations in Ca(2+)-channels during the development of diabetic cardiomyopathy. In order to examine the status of Ca2+ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of 3H-nitrendipine was studied by employing different concentrations of this Ca(2+)-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered 3H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of 3H-nitrendipine binding sites may decrease Ca(2+)-influx through voltage sensitive Ca2+ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca2+ channels may partly explain the increased sensitivity of diabetic heart to Ca2+.