Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis. NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.


A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. Using two-dimensional agarose gel electrophoresis, we determined the replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. The three sites of DNA replication initiation on the ring chromosome are specific and coincide with ARS elements. The three origins are active to different degrees; two are used > 90% of the time, whereas the third is used only 10-20% of the time. The specificity of these origins is shown by the fact that only ARS elements were competent for origin function, and deletion of one of the ARS elements removed the corresponding replication origin. The activity of the least active origin was not increased by deletion of the nearby highly active origin, demonstrating that the highly active origin does not repress function of the relatively inactive origin. Replication termination on the ring chromosome does not occur at specific sites but rather occurs over stretches of DNA ranging from 3 to 10 kb. A new region of termination was created by altering the sites of initiation. The position of the new termination site indicates that termination is not controlled by specific cis-acting DNA sequences, but rather that replication termination is determined primarily by the positions at which replication initiates. In addition, two sites on the ring chromosome were found to slow the progression of replication forks through the molecule: one is at the centromere and one at the 3' end of a yeast transposable element.


Lichen planus, liver kidney microsomal (LKM1) antibodies and hepatitis C virus antibodies. No anti-liver kidney microsomal (LKM1) antibodies were detected in 46 patients with LP, 16 of whom had also a chronic liver disease (CLD). In contrast, anti-hepatitis C virus (HCV) antibodies were found in 10% of patients with LP and in 50% of those with LP and CLD. Anti-HCV antibodies may be considered as a false-positive reaction in 56% of cases, especially when anti-LKM1 antibodies are present. Our findings do not support such a hypothesis, but suggest that CLD in LP patients is, at least in Italy, mostly a postviral chronic active hepatitis.


Fatal angioedema associated with lisinopril. To report a case of fatal angioedema associated with the use of lisinopril, a long-acting angiotensin-converting enzyme (ACE) inhibitor. Case reports, review articles, short reports, and pertinent information from the patient's medical record. Data was collected from contemporary medical journals and reviewed by both authors. Angioedema associated with ACE inhibitors (captopril and enalapril) is well documented in the literature. With increased prescribing of newer, longer-acting agents, this potentially lethal adverse reaction is of even greater concern. Because angioedema associated with ACE inhibitors is a class-related event, the number of reported cases would be expected to increase with increasing numbers of prescriptions written for these drugs. This report, describing a patient who developed angioedema following therapy with lisinopril, illustrates the severity of this adverse reaction. A 66-year-old man presented to the emergency room complaining of increased swelling of the back of his throat and difficulty breathing. Despite treatment with epinephrine, antihistamines, and corticosteroids, the patient's condition progressed from that of severe laryngeal edema to total laryngospasm and complete airway obstruction. Emergency measures to intubate the patient were complicated by severe swelling of his neck and oropharynx, forcing the physician to perform a grossly traumatic tracheotomy. The difficulty encountered during intubation deprived the patient of oxygen for a significant amount of time, precipitating cardiopulmonary arrest. The anoxic episode resulted in hypoxic, ischemic encephalopathy and, ultimately, death. Angioedema is a serious, potentially life-threatening adverse effect associated with the use of ACE inhibitors. Clinicians need to be aware of this effect when prescribing ACE inhibitors to treat hypertension and congestive heart failure, and when assessing patients presenting to the emergency room with complaints of tongue or pharyngeal swelling. Patients should be instructed to report immediately to an emergency room for medical attention if they experience any unexplained shortness of breath or swelling of the throat or tongue.


Azithromycin: the first azalide antibiotic. To discuss the chemistry, mechanism of action, spectrum of activity, pharmacokinetics, clinical trials, adverse-effect profile, drug interactions, and dosage guidelines of azithromycin, the first azalide antibiotic. Pertinent literature published between 1988 and the present was identified via a MEDLINE search. Of 77 articles retrieved, 37 have been referenced. Azithromycin is a new agent, and as such, limited data regarding this drug are available in the literature. We evaluated all pharmacokinetic, microbiologic, and basic science articles pertaining to azithromycin, and reviewed the clinical efficacy trials that we believed were of good quality for each indication for which azithromycin has received approval to date. Comparative clinical trials involving large numbers of patients, clinical outcome assessments, and recommendations for azithromycin use are included. Azithromycin is a macrolide derivative and the first of the 15-membered ring azalide class of antimicrobials. Although its mechanism of action and susceptibility to resistance are similar to those of the macrolide antibiotics, azithromycin's extended spectrum of activity includes gram-positive and gram-negative organisms, as well as atypical pathogens. Azithromycin is stable at gastric pH and has an absolute bioavailability of approximately 37 percent following oral administration. Although its serum concentrations are typically low, the drug concentrates to a high degree in tissue. Azithromycin is cleared primarily by the biliary and fecal routes; its serum half-life is in excess of 60 hours. Several clinical trials have proven that a 5-day course of azithromycin administered once a day is equally efficacious to a 7- to 14-day course of other commonly used oral antimicrobials, administered two to four times a day, for the treatment of upper and lower respiratory tract and skin and skin-structure infections. Urethritis and cervicitis caused by chlamydia are treated with a single 1-g dose. Trials have shown azithromycin's adverse-effect profile to be equal or even superior to that of other agents, with only 0.7 percent of patients discontinuing therapy versus 2.6 percent for comparable drugs. Azithromycin represents a significant improvement in the treatment of selected community-acquired infections. Although this agent may revolutionize the treatment of sexually transmitted diseases caused by chlamydia, it also should impact the management of respiratory tract and skin and skin-structure infections. Because of its unique pharmacokinetics and excellent adverse-effect profile, patient compliance should be greatly enhanced compared with other commonly used oral antimicrobials. Azithromycin's primary role in the near future will be in the community setting. Although its use in the hospital may be limited, this drug will be a convenient therapeutic option to have on hand in the emergency room and outpatient clinic. Azithromycin may also be used in the future to treat opportunistic infections in immunocompromised patients.


[Ultrastructural immunocytochemical localization of diphosphate kinase/Nm23 in human cancer cells]. Nucleoside diphosphate (NDP) kinase/Nm23 is highly expressed in certain malignant tissues, as compared with the rate found in normal or hyperplastic tissues. The potential role of this overexpression in tumor progression and the mechanisms involved in it remain to be determined. We studied the ultrastructural localisation of the NDP kinase, and in particular looked for an association of this enzyme with the microtubules or cytoplasmic membrane. Using immunocytochemical methods with an antiserum raised against NDP kinase A, we analysed tissue sections of breast carcinomas and cells in culture derived from a cervical cancer. In malignant cells, a strong labeling of the cytoplasm, related to ribosomes, was observed. No labeling of microtubules, or other intracytoplasmic components was found. No labeling of the nucleus was noted. In contrast, a strong labeling of the cytoplasmic membrane of most malignant cells was observed. In the cytoplasm of non-malignant stromal cells, a slight labeling of ribosomes was observed. These results must be taken into account with regard to the different existing hypotheses relative to the role of the NDP kinase in tumor progression, and in particular relative to its activation function on the GTP-binding proteins involved in membrane signal transduction.


[Human papillomaviruses and cellular oncogenes (c-myc, c-Ha-ras) in cutaneous and mucosal lesions in transplantation recipients]. Transplant recipients develop numerous benign and malignant cutaneous and mucosal lesions in which histological signs of human papillomavirus (HPV) infection are observed. To investigate the role of HPV and c-myc and c-Ha-ras cellular oncogenes' activation in transplanted patients lesions, we used in situ hybridization with biotinylated probes and Southern blot to detect HPV and oncogenes DNA. We analyzed 36 lesions from grafted patients: 11 common warts, 10 actinic keratoses, 13 squamous cell carcinomas and 2 anogenital papillomas. With in situ hybridization, HPV DNA was detected in 14/36 lesions, 10 of which contained several HPV types. Benign and potentially oncogenic HPV types were detected in warts as well as in squamous cell carcinomas. The Southern blot technique confirmed the distribution of HPV types. Group specific viral antigen was detected in 12 lesions, mainly warts. C-Ha-ras oncogene was amplified in 13 lesions and c-myc oncogene in 10 lesions, 9 of which showed both oncogene amplification. The results obtained with in situ hybridization for c-myc gene amplification were confirmed with the Southern blot technique in 11/14 cases. Ras and/or myc amplification was more frequent in squamous cell carcinomas and anogenital papillomas than in warts and actinic keratoses. The amplification was not always linked to the presence of HPV DNA; however, it was more frequent in lesions infected by potentially oncogenic HPV types than in lesions containing only benign HPV types. Myc and p21 oncoproteins were respectively localized in the nucleus and on the membrane of epithelial cells by immunofluorescence. Most lesions showed a good concordance between the detection of oncogene DNA and proteins. Our results suggest than c-Ha-ras and c-myc cellular oncogenes' activation and HPV infection could play a role in the cancerization of cutaneous lesions from transplant recipients.


Infectious complications associated with granulocytopenia during the treatment of poor risk or relapsed germ cell tumors. Infectious complications associated with granulocytopenia during the treatment of poor risk or relapsed germ cell tumors were studied in order to assess the frequency, morbidity and characteristics of these complications. Thirty-three cases were investigated at the Institut Gustave-Roussy; 63 episodes of granulocytopenia were observed (mean duration 7.5 days). Twenty-four (38%) of the episodes of granulocytopenia were associated with fever. In 11/24 episodes (46%), fever was associated with a documented infection. Eighteen organisms were isolated. Ten were found to be Gram-positive, 7 Gram-negative, and one Candida. The major site of infection was the lungs. One death from septicaemia occurred. In conclusion, post-chemotherapy granulocytopenia during the treatment of germ cell tumors is associated with fever in less than 50% of cases. Bacterial infection was diagnosed in only 46% of febrile neutropenic episodes and rarely appears to be associated with a fatal outcome.


Development of common cold symptoms following experimental rhinovirus infection is related to prior stressful life events. Previous studies of rhinovirus infection indicate that about one third of the persons with confirmed viral infection do not show evidence of cold symptoms. Factors that determine which infected individuals will develop colds are not known. Using a rhinovirus inoculation protocol, the authors explored the possible role of recent life events, current mood, and perceived stress in the development of symptoms in individuals known to be infected. As part of a larger study, 17 subjects were exposed to a rhinovirus and were individually isolated for 5 consecutive days; cold symptoms, mucus weights, and tissue use were monitored on a daily basis during this period. Although all 17 subjects had confirmed rhinovirus infection, only 12 subjects developed clinical colds, as indicated by self-reported symptoms and by objective symptom indices. The average number of reported major life events for the previous year was significantly higher for those who developed colds than for those who did not (p < .05). Measures of affect and perceived stress before the inoculation were not different for those who did and did not develop colds. Complementing recent research demonstrating psychosocial influences on experimental infection rates, these results provide evidence that the development of cold symptomatology in experimentally infected individuals is related to prior life events.


Mr R: a case study of withdrawal.

Mr R: a case study of withdrawal. This case study describes a patient with a long-time history of polysubstance abuse and dependence who was admitted to the hospital for detoxification from, and treatment for, his addictions. Despite physiological dependence upon the long-acting narcotic methadone, the patient did not show evidence of withdrawal from this agent until weeks after it was discontinued. This case emphasizes the psychological component of narcotic withdrawal.


Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells. The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.


Interpretation of Scatchard plots for aggregating receptor systems. Aggregation of cell surface receptors, with each other or with other membrane proteins, occurs in a variety of experimental systems. The list of systems where receptor aggregation appears to be important in understanding ligand binding and cellular responses is growing rapidly. In this paper we explore the interpretation of equilibrium binding data for aggregating receptor systems. The Scatchard plot is a widely used tool for analyzing equilibrium binding data. The shape of the Scatchard plot is often interpreted in terms of multiple noninteracting receptor populations. Such an analysis does not provide a framework for investigating the role of receptor aggregation and will be misleading if there is a relation between receptor aggregation and ligand binding. We present a general model for the equilibrium binding of a ligand with any number of aggregating receptor populations and derive theoretical expressions for observable Scatchard plot features. These can be used to test particular models and estimate model parameters. We develop particular models and apply the general results in the cases of six aggregating receptor systems where ligand binding and receptor aggregation are related: cross-linking of monovalent cell surface proteins by monoclonal antibodies, cross-linking of cell surface antibodies by bivalent ligand, antibody-induced co-cross-linking of cell surface antibodies and Fc gamma receptors, ligand-enhanced aggregation of identical epidermal growth factor receptors, aggregation of heterologous receptors for interleukin 2 to form a high-affinity receptor, and association of receptors, including those for interleukins 5 and 6, with nonbinding accessory proteins that influence receptor affinity or effector function.


Increased energy expenditure in cirrhotic patients with hepatocellular carcinoma. Basal energy expenditure was measured by indirect calorimetry in 12 cirrhotic patients with hepatocellular carcinoma. Values were compared to those observed in 12 cirrhotic patients without hepatocellular carcinoma but with similar nutrition status. Energy expenditure was also predicted in each patient by the Harris-Benedict equation. Basal energy expenditure, whether expressed as kilocalorie per day or corrected for kilogram body weight or for kilogram fat-free mass, was found increased in cirrhotic patients with hepatocellular carcinoma. These patients expended an average of 250 kcal/day more than was expected given their body size. The highest values were observed in the patients who experienced a recent significant weight loss. Our study demonstrates that the presence of hepatocellular carcinoma on liver cirrhosis increases the metabolic rate of patients. This factor could contribute to progressive malnutrition in patients with hepatocellular carcinoma and should be taken into consideration when these patients are given nutritional support.


Effect of essential fatty acids on tumor cells. An earlier study showed that essential fatty acids and their metabolites can kill tumor cells in vitro. This tumoricidal action can be correlated to an increase in generation of free radicals in the tumor cells. Evening primrose oil (EPO) is a rich source of linoleic acid and gamma-linolenic acid. We report that EPO can kill tumor cells both in vitro and in vivo. This tumoricidal action of EPO was associated with a threefold increase in superoxide generation. One of the factors that is capable of interfering with the cytotoxic action of fatty acids appears to be the protein content of the medium. Fatty acids can bind to protein and thus prevent their cytotoxic action.


Chimeric enkephalin-cystatins: opioid binding and structure-activity relationships of inhibitory domains. A chimeric inhibitor of cysteine proteinases, YGGFLQVVAGK.amide, was synthesized for use in examining SAR of its cystatin and opioid domains. Analogs were prepared by solid-phase syntheses and evaluated for inhibition of rat brain cathepsin L (E.C. 3.4.22.15) and papain (E.C.3.4.22.2), or binding to rat brain opioid receptors using tritiated DSLET (delta), DAGO (mu) and U69593 (kappa) in competitive binding assays. Cystatin consensus analogs QVVAGK- or N-Ac.QVVK.amide were weakly active as inhibitors, but were enhanced 10-fold to 20-fold with N-terminal L-, FL-, GFL-, GGFL-, or YGGFL, yielding Ki 3 microM and 26 microM for cathepsin L and papain, respectively. YGGFL itself was inactive, but expressed inhibition with N-terminal Q-, QV-, QVV-, etc. Highest activity was found with YGGFLQVK.amide (Ki, 2 microM). N-Dns analogs differentially increased inhibition for papain, whereas an N-Ac-cyclic peptide, N-Ac-CKYGGFLQVVKC.amide, was 2-fold to 10-fold less potent than the two-domain inhibitor. Chimeric peptides containing YGGFL were equipotent as delta-ligands (Ki, 1.7-3.7 nM), but were 2-fold to 10-fold more potent as mu ligands (3.7-11 nM) versus YGGFL itself (37 nM). Two analogs with -QVVAK- and -QVK.amide expressed kappa-binding properties. These data demonstrate the feasibility of using chimeric peptides as probes for exploring enzyme catalysis, and the potential for targeting inhibitors to endosomal or lysosomal compartments via receptor-mediated uptake in cells.


Indication of liver transplantation for hepatocellular carcinoma in Japan. Approximately 20,000 patients die of hepatocellular carcinoma (HCC) annually in Japan and most of them are hepatitis B virus (HBV) or hepatitis C virus (HCV) carriers. Recently, small HCC, less than 3 cm in diameter, have frequently been found by ultrasonography in the follow-up of patients with chronic liver diseases. Such cases are mainly treated by either surgical resection or percutaneous ethanol injection therapy (PEIT) with a satisfactory 5 year survival rate of 50%. In addition, the survival rate of advanced cases has gradually improved thanks to transcatheter arterial chemo-embolization combined with PEIT, radiation, hyperthermia, or immune therapy. On the other hand, our autopsy study has indicated a high frequency of extrahepatic metastasis in advanced cases. From these results, liver transplantation for HCC does not seem to be the treatment of first choice, at present, in Japan. In the future, the means to control the underlying infection of HBV or HCV as well as making an accurate imaging diagnosis for the detection of extrahepatic metastasis will become inevitably more important for successful liver transplantation in HCC.


Solution synthesis of Na+,K(+)-ATPase inhibitor-1 (SPAI-1). SPAI-1, a 49-amino acid peptide including eight Cys residues with Na+,K(+)-ATPase inhibitory activity, was synthesized by the solution procedure. Protecting groups, including the formyl group on the Trp residue, were cleaved simultaneously by HF treatment in the presence of a sufficient amount of thiol compound. After removal of the Acm group on the Cys residue, the resulting octa SH peptide was subjected to an oxidative folding reaction in the presence or absence of redox reagents and/or denaturant. Addition of redox reagents accelerated the reaction but was not crucial to the formation of the correct disulfide bonds in the molecule. The product was purified to homogeneity and found to have the same physicochemical properties and inhibitory potency as those reported for the natural product. Four intramolecularly linked disulfide bridges were assigned as connecting Cys8 to Cys37, Cys15 to Cys41, Cys24 to Cys36, and Cys30 to Cys45 based on results from a combination of enzymatic and synthetic approaches.


Spontaneous rupture of hepatocellular carcinoma: an approach with delayed hepatectomy. Two cirrhotic patients with ruptured hepatocellular carcinoma (HCC), presenting with hemoperitoneum, were successfully treated by elective hepatectomy. Both of these patients, a 67-year-old female and a 76-year-old male, had first been taken to other primary hospitals by ambulance due to hypovolemic shock. They were then found to have a mass of approximately 5 cm in the cirrhotic liver. In the initial management, however, neither any direct hemostasis by surgery nor indirect measures such as transcatheter hepatic arterial embolization were performed in either case. Instead, conservative treatment consisting mainly of fresh blood and plasma transfusions were continued for more than a month until the liver function stabilized. In both hepatectomies, the use of a microwave tissue coagulator resulted in minimal intra-operative blood loss and an appreciably excellent post-operative course. These cases point to the effectiveness of a "wait and see" policy for selected patients with ruptured HCC.


Ganciclovir therapy of congenital cytomegalovirus disease. A newborn with cytomegalovirus disease with cerebral involvement was treated with ganciclovir for 21 days. The treatment resulted in only temporary cessation of virus shedding in the urine and probably had no ameliorating effect on the long-term clinical outcome.


Bereavement follow-up service in intensive care. Following the death of a patient nurses sometimes wonder later how the surviving relatives have coped with the death of their loved one and whether there was any further help that could have been given. This report describes services available to bereaved relatives in Intensive Care Units (ICUs) throughout the UK and how, with limited resources, a formal follow-up service to all bereaved relatives can be achieved.


A novel beta-turn mimic useful for mapping the unknown topology of peptide receptors. Ethers of cis or trans D-4-hydroxyproline (Hype), adjacent to octahydroindole-carboxylic acid (Oic), introduce a beta-turn into the backbone of peptides when positioned respectively at the i+1 and i+2 position of the turn. This is confirmed by NMR experiments performed on a model tetrapeptide in water. Synthetic alteration of the ether group allows simple probing of the steric limits and electrostatic potential of a receptor binding site, a technique applied successfully to the bradykinin receptor.


Disulfide bond as peptide-resin linkage in Boc-Bzl SPPS, for potential biochemical applications. Use of disulfide bonds for labile linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin). Three bifunctional disulfide handles were synthesized for the introduction of disulfide linkage to the synthesis support. This work showed that only N-Boc aminoethyl 2-propionic acid and N-Boc aminoethyl 2-isobutyric acid were fully compatible with Boc/Bzl peptide synthesis and trifluoromethane sulfonic acid side-chain protection. Qualitative and quantitative synthesis results were comparable to those obtained by conventional peptide synthesis using polyacrylic resins. The resulting peptidyl-resins, which swelled in water or aqueous buffers, may be suitable for various biochemical applications, including use as peptide-resin conjugates for antibody production. Thiolysis by aqueous dithiothreitol released mercapto amide peptides suitable for various uses in solution (e.g., direct coupling with activated protein carrier, specific labeling in the mercapto amide group). Partial thiolysis of the disulfide linkage allowed easy post-synthetic adjustment of the peptide loading of peptidyl-resins.


Influence of the freezing-thawing-washing procedure on the cytomegalovirus DNA content of cellular blood products. There is a proven relation between cytomegalovirus (CMV) infection in seronegative immunocompromised patients and transfusion of random CMV unscreened cellular blood products (BP), where the leukocytes are thought to be the vector of transmission. We examined whether freezing, thawing, and washing, in attempt to reduce the quantity of leukocytes, also reduces the CMV-DNA-carrying cells of BP of CMV-seropositive donors and their infectivity for fibroblasts, using the polymerase chain reaction (PCR). The leukocyte contamination of the thawed-washed erythrocyte concentrates was < 1 x 10(8)/l and of the thawed-washed platelet concentrates, approximately 2.5 x 10(8)/l. All plasma samples and most samples of BPs of the CMV-seronegative controls were PCR-CMV-DNA-negative. The differences of the PCR results with the samples of CMV-seropositive donors before and after freezing were not significant. Thus we concluded that freezing-thawing-washing of cellular BP could not eliminate all CMV-DNA-bearing leukocytes. Plasma carries low risk for CMV infection, if at all.


An in vitro electrophysiological study of the colon from patients with idiopathic chronic constipation. Preparations of the circular muscle layer from the sigmoid colon resected from patients with idiopathic chronic constipation were compared, at an electrophysiological level using the sucrose-gap technique, with preparations of the same region of the intestine resected from patients with rectal carcinoma. Non-adrenergic, non-cholinergic inhibitory neuromuscular transmission, represented by inhibitory junction potentials, was present in preparations from both groups. However, the inhibitory response in preparations from constipated patients had a slower or longer time-course than in those from cancer patients. Also, rebound activity following inhibitory transmission was observed in 34% of preparations from constipated patients but was observed in 67% of preparations from cancer patients. Preparations from both groups displayed the same patterns of spontaneous activity and the same proportion of each group was quiescent. The threshold for generation of action potentials and the passive resistance of the smooth muscle membrane were the same in both groups. However, quiescent preparations from constipated patients were less likely to discharge trains of action potentials when the smooth muscle membrane was depolarized than were preparations from cancer patients. These changes in transmission processes and excitability in tissue from constipated patients are discussed in relation to altered states of colonic motility found in people with idiopathic chronic constipation.


Systems analysis of a drug dependency service in London: St George's Hospital. Elements of a systems approach to analysing service delivery are outlined. A specific example--St George's Hospital Addiction Behaviour Department--is detailed. Dynamics of the system--from the client's perspective and the workers viewpoint--demonstrate some of the ways in which emotional conflict surrounding the task is handled. The conclusions focus on what might constitute adaptive resolution of systems dilemmas as well as highlighting the public-health concerns relating to AIDS which, we argue, compromise the primary task of an addiction service at the present time.


A two-year follow-up study of alcohol dependent men rehabilitated at a special unit in a developing country. Two hundred and thirty-four alcohol dependent men who had all failed to improve after previous hospital-based interventions were admitted to an intensive 6-week residential rehabilitation programme conducted at a special unit in Sri Lanka. Outcome was assessed at 2 years post-treatment, and 36% of dependent persons remained totally abstinent. During an extended period of follow-up (mean 39.2 months) a significant association was found between abstinence and having a life partner, stable family support, a higher income and regular employment. However, there was no significant association between abstinence and age, duration of alcohol dependence, level of education or type of employment. Although the majority of alcohol dependent persons did not benefit from the programme, our results compare favourably with those of similar studies done in developed countries.


Quantitative distribution of GABA-immunopositive and -immunonegative neurons and synapses in the monkey striate cortex (area 17). The number of GABA-immunoreactive [GABA(+)] neurons and synapses was determined in functionally distinct subregions delineated as rich and poor in cytochrome oxidase (CO) in the visual cortex of adult macaque monkeys. The average numerical density (number per unit volume, Nv) of GABA(+) neurons and synapses was not significantly different between the CO-rich and -poor regions. Twenty percent of the total number of cortical neurons and 17% of the synapses were GABA(+). On average, each visual cortical neuron receives 3900 synapses, 660 of them being GABA(+). The latter were distributed on the target cell in a pattern that predicts the site of GABA influences in cortex. The major targets of GABA(+) synapses were dendritic shafts, comprising nearly two-thirds of the postsynaptic elements. About every fourth and every eighth GABA(+) synapse was devoted to dendritic spines and to neuronal somata, respectively. Axon initial segments, although the exclusive targets of GABA(+) cells, comprise less than 0.1% of structures postsynaptic to GABA(+) boutons. From this distribution, we estimate that in each cubic millimeter of striate cortex there were about 20 million GABA(+) synapses on dendritic spines, 47 million on dendritic trunks, 9 million on somata, and fewer than 0.1 million on axon initial segments. The sites of influences of GABA-immunonegative [GABA(-)] synapses were different in that they target mainly dendritic spines and dendritic trunks. About two-thirds of GABA(-) synapses were on dendritic spines, and the remainder were devoted to dendritic trunks. Only a minute fraction innervate somata. We estimate that in 1 mm3 of striate cortex there were about 235 million GABA(-) synapses on spines, 133 million on dendrites, and about 2 million on somata. The proportions of GABA(+) neurons and synapses and their target distribution did not appreciably differ from those of the visual cortex of the cat even though the numerical density of neurons was 2.5 times higher in the monkey.


Types of alcoholics: concurrent and predictive validity of some common classification schemes. This study evaluated the discriminative power and predictive validity of five common typological schemes used to classify alcoholics for theoretical or clinical purposes. A heterogenous sample of 321 alcoholics was classified according to primary vs secondary alcoholism, parental alcoholism, Fellinek's gamma-delta distinction, gender, and subtypes derived from MMPI profiles. A prospective longitudinal cohort design was employed to compare the relative ability of these typologies to differentiate alcoholics according to natural history, alcohol-related consequences, response to treatment, and post-treatment adjustment. The findings indicate that while 'one-dimensional' typologies discriminate subgroups in terms of etiological variables, presenting symptoms, and drinking patterns, none of the classification systems emerges as a strong predictor of outcome status. In addition, these typologies do not discriminate well with respect to the alcoholic's drinking patterns and presenting symptoms, except in areas closely connected with the model (e.g. alcoholics with antisocial personality (ASP) indicate more social problems related to alcohol use than do primary alcoholics). Because there is a great deal of overlap among certain subtypes identified within different typological models (e.g. alcoholics with primary ASP tended to have a positive family history and a gamma pattern of impaired control), we hypothesize that empirical clustering techniques that search for naturally occurring commonalities among alcoholics may be a better way to identify homogeneous subtypes.


What happens to drinking after therapeutic intervention? Eighty-four men and 51 women who had been recruited to treatment through newspaper advertisements were matched pairwise and randomly assigned to either brief counselling or short-term group therapy. All participants were followed-up after 3, 9, 15 and 21 months by personal interviews and medical examinations. The average reduction in alcohol consumption was greatest to start with in the group attending group therapy, but this group experienced greater set-back during the following period. At 21 months there were no significant differences between the two groups as regards amount of alcohol consumed, GGT-levels, deaths or number of alcohol-related hospitalizations. The sample as a whole maintained a significant reduction in alcohol consumption throughout the follow-up period. At 21 months half of the sample had reduced their consumption with more than 50%, one-fourth had reduced 20-50%, one-tenth changed less than 20% and only 18% of the sample had increased their consumption with more than 20% compared with their baseline consumption. The reductions took place mainly by way of less frequent drinking, fewer episodes of heavy drinking and reduced consumption during weeks of moderate drinking. When heavy drinking episodes did occur, there were only minor changes in the amounts consumed.


Sodium channel gene defects in the periodic paralyses. Abnormal Na+ currents that produce membrane depolarization have been associated with the episodes of muscle weakness that are the hallmark of the periodic paralyses. There is now strong evidence that various point mutations in the gene encoding the adult skeletal muscle voltage-dependent Na+ channel produce these abnormal currents, and are responsible for the expression of the disease phenotype.


A method for the calculation of protein alpha-CH chemical shifts. The chemical shifts of C(alpha)H protons have been calculated for 9 proteins, based on coordinates taken from high-resolution crystal structures. Chemical shifts were calculated using ring-current shifts, shifts arising from magnetic anisotropies of bonds, and shifts arising from the polarizing effect of polar atoms on the C(alpha)H bond. The parameters used were refined iteratively to give the best fit to (experimental-random coil) shifts over the set of 9 proteins. A further small correction was made to the averaged Gly C(alpha)H shift. The calculated shifts match observed shifts with correlation coefficients varying between 0.45 and 0.86, with a standard deviation of about 0.3 ppm. The differences between calculated and observed shifts have been studied in detail, including an analysis of different crystal structures of the same protein, and indicate that most of the differences can be accounted for by small differences between the structure in solution and in the crystal. Calculations using NMR-derived structures give a poor fit. The calculations reproduce the experimentally observed differences between chemical shifts for C(alpha)H in alpha-helix and beta-sheet. Most of the differentiation in secondary-structure-dependent shifts arises from electric field effects, although magnetic anisotropy also makes a large contribution to the net shift. Applications of the calculations to assignment (including stereospecific assignment) and structure determination are discussed.


1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids. Two-dimensional (2D) 1H NMR experiments using deuterium labeling have been carried out to investigate the solution of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), which consists of 155 amino acids. To simplify the 1H NMR spectra, two fully deuterated enzymes bearing several protonated amino acids were prepared from an RNase HI overproducing strain of E. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, Ile, Val, and Leu. The other was labeled by only protonated His and Ile. The 2D 1H NMR spectra of these deuterated RNase HI proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.


Selenoproteins in mitochondria and cytosol of Saccharomyces uvarum after growth in sodium selenite-supplemented media. The extent of the biosynthesis of selenoproteins in mitochondria and cytosol of Saccharomyces uvarum depends on the sodium selenite concentration in the medium. In mitochondria there is a selenoprotein (SP 1) exhibiting glutathione peroxidase activity whose concentration already reaches a maximum at low concentrations of sodium selenite. A second selenoprotein (SP 2) was found in mitochondria and cytosol. Both proteins contain L-selenocysteine. The molecular masses of SP 1 and SP 2 are 73,000 Da and 83,000 Da, respectively. A subunit of SP 1 was found to have a molecular mass of 30,000 Da. SP 2, identified as a glycoprotein, has subunits with molecular masses of 36,500 Da and 5,000 Da. The selenium concentration of the total yeast increases from 260 micrograms/kg dry weight to 280 mg/kg dry weight after supplementation of the medium with sodium selenite.


Scheduling of monoaminergic neurotransmitter receptor expression in the primate neocortex during postnatal development. Quantitative in vitro autoradiography was used to study the postnatal development of monoaminergic receptors (D1 and D2 dopaminergic, 5-HT1 and 5-HT2 serotonergic, and alpha 1, alpha 2, and beta noradrenergic sites) in the prefrontal, primary motor, somatosensory, and visual cortex of rhesus monkeys at birth and 1, 2, 4, 8, 12, 36, and 60 months of age. The density of all receptors studied increased rapidly within the first 2 postnatal months to levels as high as two times that recorded in the adults. After the fourth month, receptor density began a decline that subsided around the time of puberty. This course of developmental change was similar in all cortical layers and in all regions examined. However, the magnitude of the transient overproduction and eventual reduction in receptor density varied across the cortical layers and cytoarchitectonic areas in a manner specific to the individual receptor sites. Overall, cortical maturation was associated with the increased tendency of monoaminergic receptors to concentrate preferentially in the superficial cortical layers. The common developmental course of monoaminergic receptors in diverse cytoarchitectonic areas reveals an impressive coordination in the expression and regulation of these functionally relevant proteins in the cerebral cortex during infancy and adolescence.


Purification of cytochrome c oxidase by lysine-affinity chromatography. A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.


Heterologous expression of recombinant human glutathione transferase A1-1 from a hepatoma cell line. A cDNA clone, lambda GTHA1, encoding human glutathione transferase A1-1 has been isolated from a hepatoma HepG2 cDNA library. At the nucleotide level, the new clone showed minor differences from cDNA deriving from normal liver, but the deduced amino acid sequence was identical to the structure previously described. The protein was expressed from a plasmid, pKHA1, and isolated by a single-step affinity purification on an S-hexylglutathione Sepharose matrix. The yield of the recombinant protein was 165 mg from a 3-liter culture of bacteria.


High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase. The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.


Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli. The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique. Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant enzyme has good activity (approximately 30 nkat/mg).


Effects of a 5-lipoxygenase inhibitor, AA861, on lipoxygenase metabolism and superoxide anion generation by human polymorphonuclear leukocytes--potentiation of superoxide anion generation by LTB4. We studied the influence of a selective 5-lipoxygenase inhibitor, AA861, on the generation of the superoxide anion (O2-) and the lipoxygenase metabolites by human polymorphonuclear leukocytes (PMN). PMN produce O2- in a dose-dependent manner following stimulation with arachidonic acid (AA), leukotriene B4 (LTB4), or C5a. When PMN were stimulated with one of those three agents in the presence of high doses of AA861 (1-10 micrograms/ml), a significant reduction of O2- release was observed. In contrast, the generation of O2- by PMN stimulated by LTB4 was potentiated at lower concentrations of AA861 (0.025-0.25 micrograms/ml). However, stimulation with AA or C5a did not influence O2- generation in the presence of AA861 at the same concentration range. Furthermore, treating the PMN with the cyclooxygenase inhibitor, acetylsalicylic acid, did not potentiate the generation of O2- by stimulation with LTB4 over a wide range of concentrations. Quantification of lipoxygenase metabolites by reverse-phase high-performance liquid chromatography revealed that a high concentration of AA861 (0.5-5 micrograms/ml) completely inhibited the production of LTB4 and its omega-oxidative metabolites by PMN following stimulation with 100 microM AA, but only partially inhibited that of 5-hydroxyeicosatetraenoic acid (5-HETE). AA861 at a concentration of 5 micrograms/ml significantly increased the production of 15-HETE by PMN following the same stimulation. AA861 did not influence catabolism of LTB4 added to the reaction mixture to its omega-oxidative products by PMN over a wide range of concentrations. These findings suggest that the inhibition of 5-lipoxygenase metabolism may stimulate 15-lipoxygenase in human PMN.(ABSTRACT TRUNCATED AT 250 WORDS)


[Prevalence of Chlamydia trachomatis in genital samples from Abidjan]. A study of direct genital swabs achieved in Abidjan, on 116 men and 131 women consulting for urogenital complaints, has revealed that the men show a prevalence of 28.4% Chlamydia trachomatis, and of 18.1% Neisseria gonorrhoeae. Concerning the women the prevalence of the same germs are 13.7% for Chlamydia trachomatis, and 4.6% for Neisseria gonorrhoeae. These results show the importance of Chlamydia trachomatis as a sexually transmitted disease in Abidjan (Côte-d'Ivoire). No differences were observed according to age in the two groups.


Second messenger systems stimulated by bradykinin in osteoblastic cells: evidence for B2 receptors. The effects of bradykinin, analogs and inhibitors on the human osteoblastic osteosarcoma cell lines Saos-2 and G292 and on normal rat calvarial osteoblastic cells were investigated. In all cell types, bradykinin (1 nM-100 microM) caused significant time- and dose-dependent changes in the levels of inositol phosphates. Neomycin inhibited the inositol phosphate response to bradykinin, while indomethacin had no effect. Bradykinin also elicited a dose-dependent increase in free cytosolic calcium concentration. Bradykinin and T-kinin did not affect cyclic AMP levels in these cells. Doses of des-Arg9-bradykinin, a B1 receptor agonist, up to 100 nM did not stimulate the osteoblastic inositol phosphate response. In addition, the bradykinin-stimulated inositol phosphate response was unaffected by des-Arg9-[Leu8]-bradykinin, a B1 receptor antagonist, while it was inhibited by D-Arg-[Hyp3-[beta-(2-thienyl)-Ala]5,8-D-Phe7]-bradykinin, a B2 receptor antagonist. These results suggest that in osteoblastic cells the mechanism of action of bradykinin involves stimulation of the phosphoinositide metabolism and increases in cytosolic calcium levels through activation of B2 receptors.


Conversion relations for quantitative CT bone mineral densities measured with solid and liquid calibration standards. A vast data base exists for QCT measurements of bone mineral density (BMD) referenced to K2HPO4 in water (liquid) standards. To effectively utilize the more stable hydroxyapatite in water-equivalent plastic (solid) standards that have recently been introduced, it will be necessary to derive conversion relations. A study was performed to investigate the dependence of these relations upon x-ray tube voltage, marrow composition, and patient body size. Test objects included five diverse composition vertebral marrow inserts within three different size lumbar simulators and an L1 vertebra within a Humanoid phantom. The calibration standards were manufactured by Image Analysis, and all data were acquired with a GE 9800 CT scanner operated at 80 kVp and 140 kVp. Least square fits to corresponding liquid versus solid referenced BMD measurements of the inserts all had r's > 0.999. SEEs, < 2 mg/ml, and intercepts of approximately 0. The slopes (BMDK2HPO4/BMDhydroxyapatite) for the various body sizes were all about the same with values of 0.86, 0.81, and 0.96 to 1.02 for the single-energy@80 kVp, single-energy@140 kVp, and dual-energy measurements, respectively. Corresponding ratios for the Humanoid vertebra were 0.86, 0.82, and 0.96. The conversion relations were essentially independent of marrow composition and body size but did depend upon kVp. Finally, although the solid standards are more stable, they may still exhibit problems, and these are discussed.


Computer system for assisting with clinical interpretation of tumour marker data. To design and evaluate a computer advisory system for the treatment of gestational trophoblastic tumour. A comparison of clinicians' treatment decisions with those of the computer system. Two datasets were used: one to calibrate the system and one to independently evaluate it. Department of medical oncology. Computerised records of 290 patients with low risk gestational trophoblastic tumour for whom the advisory system could predict the adequacy of treatment. The calibration set comprised patients admitted during 1979-86(227) and the test set patients during 1986-89(63). The system's accuracy in predicting need to change treatment compared with clinicians' actions. The mean time faster that the system was in predicting the need to change treatment. On the calibration dataset the system was 94% (164/174) accurate in predicting patients whose treatment was adequate, recommending change when none occurred in only 10 (6%) patients. In patients whose treatment was changed the system recommended change earlier than clinicians in 39/53 cases (74%), with a mean time advantage of 14.9 (SE 2.02) days. On the test dataset the system had an accuracy of 91% (31/34) in predicting treatment adequacy and a false positive rate of 9% (3/34). The system recommended change earlier than clinicians in 22/29 cases (76%), with a mean time advantage of 12.5 (2.22) days. The computer advisory system could improve patient management by reducing the time spent receiving ineffective treatment. This has implications for both patient time and clinical costs.


Histopathology of malignant salivary gland tumours. This report is based upon the Salivary Gland Register in Hamburg and on the second revised edition of the WHO Histological Typing of Salivary Gland Tumours. The group of malignant salivary gland tumours contains carcinomas, malignant non-epithelial tumours, malignant lymphomas and secondary tumours. The various carcinomas are classified in a continuous separate listing because the different types are distinguished not only by histopathology, but also by differences in prognosis and treatment. The term "tumour" is replaced by "carcinoma" in two entities: acinic cell carcinoma and mucoepidermoid carcinoma. New entities are: polymorphous low-grade adenocarcinoma, basal cell adenocarcinoma, salivary duct carcinoma and malignant myoepithelioma. Carcinoma in pleomorphic adenoma can be distinguished as non-invasive and invasive carcinoma, and carcinosarcoma. Malignant non-epithelial tumours are mostly malignant fibrous histiocytoma, malignant schwannoma and rhabdomyosarcoma. The large majority of malignant lymphomas are non-Hodgkin-lymphomas with high differentiation. Many lymphomas are associated with chronic immunosialadenitis (Sjögren's syndrome). Secondary tumours are mostly metastases from primary squamous cell carcinomas or from melanomas of the skin (head and neck area). Haematogeneous metastases are very rare (mainly from lung, kidney or breast).


[The neurotropic and cardiotropic properties of new amino acid derivatives of isonicotinic acid]. Different derivatives of isonicotinic acid are used widely enough as antimicrobial and antituberculous agents. However, their neurotropic and cardiotropic effects have been studied little. The paper is concerned with investigations of these types of the activity of the new derivatives of isonicotinic acid: beta-phenyl-beta-alanine, l-proline, DL-valine, beta-alanine and DL-threonine synthesized for the first time at the Institute of Fine Organic Chemistry, Academy of Sciences of Armenia.


[Benzodiazepine receptors of the neurons and astrocytes]. Ultrastructural investigations of different parts of rat brain after diazepam administration revealed benzodiazepine receptors on the neuronal surface and plasma membrane of astrocytes of the sensorimotor cortex, hippocampus and central grey substance. Subsuperficial cisternae that replenish deficit of plasma membrane required for coated vesicles formation were only seen in the neurons and were absent from the astrocytes. The fate of internalized diazepam in the nerve and glial cells were different: in the neurons, ligand is destroyed in lysosomes and multivesicular bodies, while in the astrocytes, exceptionally in lysosomes.


Oncogenes and tumour-suppressor genes in squamous cell carcinoma of the head and neck. Cancer is now considered to be a multi-hit process which involves a number of aberrant genetic events culminating in malignant transformation. In squamous cell carcinoma (SCC) of the head and neck the action of both oncogenes and tumour-suppressor genes has been identified during the course of the disease. Cytogenetic analysis of these carcinomas has demonstrated chromosomal breakpoints, particularly in the regions of 1p22 and 11q13 together with frequent amplification of the proto-oncogenes in the 11q13 amplicon; int-2, hst-1 and bcl-1. Ras mutations have been infrequently identified in the Western World whereas ras over-expression has been a common finding and may be associated with the early development of head and neck cancer. C-myc over-expression appears to correlate with a poor prognosis for these patients. The tumour-suppressor gene p53 is also thought to be involved in the development of SCC in head and neck tumours and its aberrant expression is associated with a history of heavy smoking and heavy drinking. E-cadherin, a putative tumour-suppressor gene is down-regulated in poorly differentiated head and neck SCC and maybe important in nodal metastasis. A recent study has indicated that the Human Papilloma Virus (HPV 16 and 33) has a role in the aetiology of tonsillar carcinomas and HPV has been shown to produce transforming proteins which bind to and inactivate the p53 tumour suppressor gene. This evidence suggests that the possibility of a viral mechanism for the development of SCC in the head and neck should be considered. This paper proposes a series of genetic events to explain the development of SCC of the head and neck.


Pathology of the liver in mucopolysaccharidosis: light and electron microscopic assessment before and after bone marrow transplantation. Serial liver biopsies were obtained in 20 patients undergoing bone marrow transplantation (BMT) for mucopolysaccharidosis (MPS). The 13 patients with MPS I, one with MPS II, four with MPS III, and two with MPS VI underwent liver biopsy prior to and from 1 to 37 months after BMT. The amount of accumulated glycosaminoglycan (GAG) was assessed by semiquantitation of Kupffer cell staining with colloidal iron and by counting the number of hepatocellular GAG-containing lysosomes in electron micrographs. Eleven of 13 patients with MPS I achieved engraftment, and 10 of the 11 cleared the Kupffer cells and hepatocytes of GAG by 3 to 19 months post-BMT. Two patients with autologous recovery demonstrated persistent hepatocyte inclusions. The three patients with MPS II and MPS VI engrafted and showed clearance of hepatocyte and Kupffer cell GAG by 7 months after BMT. All four patients with MPS III engrafted. Although the Kupffer cells in these patients were cleared of GAG by 12 months after BMT, hepatocellular inclusions persisted in all four. For MPS I, II and VI, donor engraftment was associated with resolution of lysosomal storage material in donor-derived Kupffer cells and untransplanted hepatocytes, indicative of transcellular metabolic correction. Failure of hepatocyte clearance in one case of MPS I and all patients with MPS III suggested a diminished capacity of the graft-derived enzyme to enter the hepatocyte lysosomes in these patients.


Inflammatory peripheral neuropathy following high dose chemotherapy and autologous bone marrow transplantation. A 40-year-old man with non-Hodgkin's lymphoma developed severe ascending sensorimotor neuropathy 10 days after treatment with high dose chemotherapy and autologous bone marrow rescue. The neuropathy had axonal plus demyelinating features on electrophysiological studies. Sural nerve biopsy showed heavy infiltration of the epineurium and endoneurium with mononuclear cells. The patient had no other evidence of graft-versus-host disease. He failed to respond to plasmapheresis but responded to high dose steroids.


High-dose chemotherapy with autologous bone marrow transplantation in the treatment of high grade astrocytomas in adults: therapeutic rationale and clinical experience. High-grade astrocytomas are the most common primary malignant brain tumors in adults and constitute a significant cancer problem in the USA accounting for nearly 11,000 deaths annually. Despite improvements in neurosurgical techniques and radiotherapy, the prognosis of these patients remains dismal with median survivals of less than 1 year. Recent data suggest that conventional chemotherapy may be more active in these tumors than previously appreciated but survival has not been substantially altered. Poor drug delivery secondary to the unique neuroanatomical and pathologic constraints of the blood-brain and blood-tumor barrier may contribute to the lack of a significant impact of conventional dose chemotherapy on the natural history of these tumors. In addition, the substantial intratumor cellular heterogeneity invariably found in these tumors undoubtedly contributes to the development of drug resistance. Based on trials of high-dose chemotherapy with autologous bone marrow transplantation (ABMT) in other tumor types, clinical investigators have begun to consider the potential for high-dose chemotherapy to overcome problems of poor drug delivery and cellular drug resistance in brain tumors. In this report, the rationale behind the use of high-dose chemotherapy with ABMT in the treatment of patients with high-grade astrocytomas, and relevant clinical trials conducted to date, are reviewed. Although these trials suggest that survival is not improved in patients treated at the time of relapse, early data suggest that high-dose chemotherapy with ABMT may hold promise as adjuvant treatment in this patient population.


Suramin inhibits excitatory junction potentials in guinea-pig isolated vas deferens. 1. Intracellular microelectrode recording techniques were used to investigate the action of the putative P2-purinoceptor antagonist, suramin, on sympathetic neurotransmission in the guinea-pig isolated vas deferens. 2. The resting membrane potential of the control cells was 67.4 +/- 0.7 mV (n = 48). Field stimulation of the sympathetic nerves innervating the vas deferens produced excitatory junction potentials (e.j.ps) which reached a mean magnitude of 8.5 +/- 0.8 mV (n = 23) when fully facilitated at a stimulation frequency of 0.5 Hz. 3. Introduction of suramin 1-100 microM produced no change in the resting membrane potential of the smooth muscle cells, but gradually reduced e.j.p. magnitude. Suramin, 20 microM, reduced the mean magnitude of the fully facilitated e.j.ps to 1.4 +/- 0.3 mV (n = 18). 4. After suramin-induced inhibition of e.j.ps, nerve stimulation at 1-8 Hz resulted in summation of e.j.ps to a subthreshold level. Subsequent introduction of the alpha-adrenoceptor antagonists, prazosin or phentolamine (1 microM) did not reduce the magnitude of the summated e.j.ps. 5. The results support the proposal that e.j.ps in vas deferens are mediated by adenosine 5'-triphosphate, and not by noradrenaline, and confirm that suramin can antagonize responses mediated via P2-purinoceptors.


Vasopressin-stimulated [3H]-inositol phosphate and [3H]-phosphatidylbutanol accumulation in A10 vascular smooth muscle cells. 1. The characteristics of vasopressin-stimulated phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and phosphatidylcholine (PtdCh) hydrolysis were examined in A10 vascular smooth muscle cells (VSMC), by assessing the formation of [3H]-inositol phosphates ([3H]-IP) and the accumulation of the phospholipase D (PLD) specific product, [3H]-phosphatidylbutanol ([3H]-PtdBuOH). 2. Vasopressin ([Arg8]-VP) and a number of related analogues stimulated the accumulation of [3H]-IP and [3H]-PtdBuOH with similar EC50 values, generating the same rank order of potency for each response (Arg8-VP = vasotocin = Lys8-VP much greater than oxytocin). 3. Inhibition of vasopressin-stimulated [3H]-IP and [3H]-PtdBuOH accumulation by the V1a receptor antagonists, Des-Gly9[beta-mercapto-beta,beta,-cyclopentamethylene propionyl, O-Et-Tyr2,Val4,Arg8]-vasopressin generated similar IC50 values suggesting that both these responses are mediated through the activation of a single V1a receptor subtype. 4. The onset of vasopressin-stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) mass formation preceded [3H]-PtdBuOH accumulation indicating that PtdCh hydrolysis was activated subsequent to PtdIns(4,5)P2 breakdown. 5. The protein kinase C (PKC) activator, tetradecanoylphorbol acetate (TPA) also stimulated [3H]-PtdBuOH accumulation. Preincubation with the PKC inhibitor Ro-31-8220 abolished both TPA- and vasopressin-stimulated [3H]-PtdBuOH, suggesting that the intermediate activation of protein kinase C is involved in the regulation of PLD by vasopressin. 6. Pretreatment of the A10 VSMC with Ro-31-8220 (100 microM) also potentiated vasopressin-stimulated Ins(1,4,5)P3 mass formation.Therefore stimulation of PKC may have opposing roles in the regulation of agonist activation of PLC and PLD.7. Preincubation of the cells with EGTA, verapamil, or the receptor-operated calcium channel antagonist, SK&F 96365, reduced vasopressin-stimulated [3H]-PtdBuOH accumulation by approximately 30%, suggesting that influx of calcium has a significant role to play in the regulation of vasopressinstimulated PLD activity.


Pharmacological characterization of adenosine A1 and A2 receptors in the bladder: evidence for a modulatory adenosine tone regulating non-adrenergic non-cholinergic neurotransmission. 1. The nerve-evoked contractions elicited by transmural electrical stimulation of mouse urinary bladders superfused in modified Krebs Ringer buffer containing 1 microM atropine plus 3.4 microM guanethidine were inhibited by adenosine (ADO) and related nucleoside analogues with the following rank order of potency: R-phenylisopropyladenosine (R-PIA) greater than cyclohexyladenosine (CHA) greater than 5'N-ethylcarboxamido adenosine (NECA) greater than ADO greater than S-phenylisopropyladenosine (S-PIA). Tissue preincubation with 8-phenyltheophylline (8-PT) displaced to the right, in a parallel fashion, the NECA concentration-response curve. 2. The contractions elicited by application of exogenous adenosine 5'-triphosphate (ATP) were also inhibited by ADO and related structural analogues. The rank order of potency to reduce the motor response to ATP was: NECA greater than 2-chloroadenosine (CADO) greater than R-PIA greater than ADO greater than CHA greater than S-PIA. 3. The ADO-induced ATP antagonism was of a non-competitive nature and was not specific. Tissue incubation with 10 microM NECA not only reduced the motor responses elicited by ATP, but also 5-hydroxytryptamine, acetylcholine and prostaglandin F2 alpha. The action of NECA was antagonized following tissue preincubation with 8-PT. The inhibitory action of NECA was not mimicked by 10 microM CHA. 4. The maximal bladder ATP contractile response was significantly increased by tissue preincubation with 5-30 microM 8-PT. 5. The 0.15 Hz evoked muscular twitch was significantly increased by 8-PT while dipyridamole consistently reduced the magnitude of the twitch response. These results are consonant with the hypothesis that an endogenous ADO tone modulates the bladder neurotransmission. 6. A working model is proposed suggesting the presence of ADO-Al and A2 receptors in the mouse urinary bladder. The A1 receptor subpopulation is probably of presynaptic origin whereas the smooth muscle membranes contain a population of the A2 receptor subtype.


Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca(2+)-pump, reduces Ca(2+)-dependent K+ currents in guinea-pig smooth muscle cells. 1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca(2+)-ATPase in sarcoplasmic reticulum (SR), on membrane ionic currents were examined in single smooth muscle cells freshly isolated from ileal longitudinal strips and urinary bladder of the guinea-pig. 2. Under whole-cell clamp, CPA (1-10 microM) reduced peak outward current elicited by depolarization in a concentration-dependent manner. The concentration of CPA required for 50% decrease in the peak outward current was approximately 3 microM in ileal cells under these conditions. The current reduced by CPA recovered by more than 70% after washout. 3. The transient outward current elicited by application of 5 mM caffeine at a holding potential of -50 mV in Ca2+ free solution was almost abolished, when the preceding Ca(2+)-loading of the cell in a solution containing 2.2 mM Ca2+ was performed in the presence of 3 microM CPA. 4. When the Ca(2+)-dependent K+ current (IK-Ca) and Ca2+ current (ICa) were inhibited by addition of Ca2+, the remaining delayed rectifier type K+ current was not affected by 10 microM CPA. When outward currents were blocked by replacement of K+ by Cs+ in the pipette solution, the remaining ICa was not affected by 10 microM CPA. 5. CPA (10 microM) did not affect the conductance of single maxi Ca(2+)-dependent K+ channels or the Cd(2+)-dependence of their open probability in both inside- and outside-out configurations. 6. These results indicate that IK-Ca is selectively and strongly suppressed by CPA.Its effects may be attributed to a decrease in Ca2"-uptake into SR, resulting in a decrease in Ca2"-induced Ca24 release which is triggered by Ca24 entering through voltage-dependent Ca24 channels and therefore less activation of these K channels.7. CPA may be extremely valuable pharmacological tool for investigating intracellular Ca24 mobilization and ionic currents regulated by intracellular Ca24.


GH4ZD10 cells expressing rat 5-HT1A receptors coupled to adenylyl cyclase are a model for the postsynaptic receptors in the rat hippocampus. 1. Vasoactive intestinal polypeptide (VIP) stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) production by cultured GH4ZD10 cells with an EC50 value of about 7 nM. The extracellularly recovered cyclic AMP predominated, and was reduced by co-incubation with 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) and 5-hydroxytryptamine (5-HT), whereas dopamine (0.1-30 microM) did not reduce VIP-stimulated cyclic AMP production. 2. The responses to 5-HT and 8-OH-DPAT were blocked by (-)-alprenolol and NAN 190. The antagonism by (-)-alprenolol was competitive in nature with a pA2 value of 7.0. 3. The responsiveness of the cells to 5-HT agonists was highly dependent upon the culturing conditions used. Thus, 8-OH-DPAT inhibition of VIP (30 nM)-stimulated cyclic AMP production decreased with increasing passage number of the cells. Reduction of the zinc concentration used to promote expression of the 5-HT1A receptor gene produced a greater sensitivity of the cells to 5-HT agonists. 4. Under such conditions, the following efficacies (5-HT = 100) were found: lisuride 106, (+)-lysergic-acid diethylamide 100, 5-methoxy-N,N-dimethyltryptamine 98, RU 24949 98, 5-carboxamidotryptamine 97, (+/-)-8-OH-DPAT 90, (+)-8-OH-DPAT 87, 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine 86, flesinoxan 79/88, (-)-8-OH-DPAT 62, buspirone 43/50, ipsapirone 46. Spiroxatrine and spiperone had a low intrinsic activity, but reduced the response to 5-HT. These efficacies are similar to those reported in the literature for post-synaptically localized 5-HT1A receptors in the rat hippocampus. Thus, the GH4ZD10 cells serve as a useful in vitro model system for these receptors.


H3 receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450. 1. Thioperamide (TP), an imidazole and a highly potent, specific antagonist of the histamine H3 receptor, inhibited the secretion of cortisol from bovine isolated adrenocortical cells (IC50 0.20 microM) and in the rat (5 mg kg-1) prevented both basal and stress-induced secretion of corticosterone. 2. In adrenocortical microsomes, low affinity binding of [3H]-histamine (KD 27.7 microM) was potently inhibited by TP (Ki 0.33 microM). 3. In adrenocortical microsomal membranes, both histamine and TP yielded type II difference absorption spectra, characteristic of the interaction between imidazole and cytochrome P450 enzymes. Dissociation constants for binding to P450, calculated from spectral data, were 15.9 microM and 1.5 mM for histamine, and 0.3 microM and 3.7 microM for TP. 4. In view of previously reported evidence for an intracellular mediator role of histamine in platelets, the present findings suggest a physiological role for histamine in the modulation of adrenal P450 monooxygenases that generate adrenocortical steroids. 5. The results suggest that direct adrenocortical inhibition by thioperamide at a non-H3 intracellular site must be taken into account in studies designed to elucidate functional roles of H3 receptors.


Pharmacodynamics of the anticonvulsant effect of oxazepam in aging BN/BiRij rats. 1. The purpose of this investigation was to examine the influence of increasing age on the pharmacokinetics and the time course of the anticonvulsant response of oxazepam in BN/BiRij rats as an animal model of aging. 2. Oxazepam was administered intravenously in a dose of 12 mg kg-1 body weight and the anticonvulsant effect intensity was measured as elevation above baseline of a threshold for induction of localized seizure activity (TLS). Direct cortical stimulation with ramp shaped electrical pulse trains of increasing intensity was used to determine this threshold. 3. The pharmacological effect vs. time profile showed in young rats an anticonvulsant component followed by proconvulsant component which is suggestive for the occurrence of acute tolerance and/or withdrawal syndrome. With increasing age the proconvulsant component disappeared, resulting in a monophasic effect profile (anticonvulsant effect only) at the age of 35 months with significantly higher anticonvulsant effect intensity immediately following drug administration. No age-related changes in the pharmacokinetic parameters of oxazepam were observed. 4. In five animals of each age group, benzodiazepine receptor binding characteristics were determined in vitro with [3H]-flunitrazepam as a ligand. Both receptor density and affinity did not show age-related changes. Available literature data on post-receptor events do not indicate conclusive age-related changes. 5. It is concluded, that the observed change in the pharmacodynamics of anticonvulsant effect of oxazepam can be explained by the disappearance of the tolerance/withdrawal phenomenon. This is compatible with a decreased efficiency of homeostatic control mechanisms in the elderly.


Cardiac alpha 1-adrenoceptor densities in different mammalian species. 1. alpha 1-Adrenoceptor densities were studied in cardiac membrane preparations from several mammalian species including human failing hearts under identical experiment conditions; the alpha 1-adrenoceptor antagonist, [3H]-prazosin, was used as radioligand. End-stage heart failure (NYHA IV) in human hearts was due to idiopathic dilated cardiomyopathy. 2. The ventricular alpha 1-adrenoceptor densities were not significantly different in guinea-pig, mouse, pig, calf, and man (11 to 18 fmol mg-1 protein) but about 5 to 8 fold smaller than in rat (about 90 fmol mg-1 protein). Right and left ventricular receptor densities were similar in these species. 3. A sufficient amount of right and left atrial tissue was obtained from rabbit, pig, calf, and man only. The alpha 1-adrenoceptor densities in both atria of these species were found to be at the detection limit of the method used (less than 8 fmol mg-1 protein). 4. The equilibrium dissociation constant (KD) was similar in all species studied ranging from 0.047 +/- 0.006 to 0.063 +/- 0.007 nmol l-1. 5. It is concluded that differences in alpha 1-adrenoceptor density between atria and ventricles may exist in mammalian species. The exceptionally high density of these receptors in rat ventricles seem to be a particular feature in these animals.


Activation of the human neutrophil 5-lipoxygenase by leukotriene B4. 1. In the present study, we demonstrate that leukotriene B4 (LTB4) has the ability to activate the human neutrophil 5-lipoxygenase (5-LO). 2. Stimulation of neutrophils with 30 nM 14,15-dideuterio-LTB4 (D2-LTB4) failed to induce the synthesis of LTB4 from endogenous arachidonic acid (AA), but stimulated the formation of LTB4 from 3.3 microM exogenous AA, as determined by GC-MS analysis. 3. The stimulatory effect of LTB4 on 5-LO activity was further examined with an alternative substrate; LTB4 time- and dose-dependently stimulated the 5-LO-mediated conversion of exogenous 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoate (15-HpETE) into 5(S),15(S)-dihydroxy-6,8,11,13,-(E,Z,Z,E)-eicosatetraenoate (5,15-DiHETE), with a threshold effect at 300 pM. 4. The ability of LTB4 to activate the 5-LO showed structural specificity, since LTB4 was found to be 100 times more potent than omega-hydroxy-LTB4, and 300 times more potent than its delta 6-trans-12-epi-isomer. 5. The LTB4-induced 5-LO activation was effectively inhibited by MK-886 (an inhibitor of 5-LO translocation), by pertussis toxin, and by the LTB4 receptor antagonist, LY-223982. 6. These results demonstrate that the binding of LTB4 to its cell-surface receptor results in 5-LO activation in a process mediated by pertussis toxin-sensitive guanine nucleotide-binding proteins. Our data also suggest that the underlying mechanism involves a translocation of the 5-LO to the membrane. These findings raise the possibility that LTB4 produced by phagocytes may positively feedback on its own synthesis.


Cardiovascular effects of substituted tetrahydroisoquinolines in rats. 1. A series of substituted tetrahydroisoquinolins derived from the cleavage products of tetrandrine were found to inhibit [3H]-nitrendipine binding to rat cerebral cortical membranes. Those compounds which displaced [3H]-nitrendipine binding were also able to inhibit high KCl-induced contraction of rat aorta in vitro. 2. There was a significant correlation between the ability of these tetrahydroisoquinolines to inhibit [3H]-nitrendipine binding and KCl-induced contraction (r = 0.99, P less than 0.001). 3. CPU-23 (1-(1-[(6-methoxy)-naphth-2-yl])-propyl-2-(1-piperidine)-acetyl- 6,7- dimethoxy-1,2,3,4-tetrahydroisoquinoline), one of the most potent compounds identified in this series, behaved as a simple competitive inhibitor at the [3H]-nitrendipine binding site and reduced the apparent affinity but not the maximal number of binding sites in saturation analysis. 4. In contrast to nifedipine which caused hypotension and tachycardia, CPU-23 induced both hypotension and bradycardia in a dose-dependent manner in pentobarbitone-anaesthetized Sprague-Dawley rats, spontaneously hypertensive and age-matched normotensive WKY rats. 5. It is suggested that CPU-23 may exert its cardiovascular effects via interaction with the dihydropyridine binding site on the L-type calcium channel.


The role of complement, platelet-activating factor and leukotriene B4 in a reversed passive Arthus reaction. 1. The mechanisms underlying oedema formation induced in a reversed passive Arthus (RPA) reaction and, for comparison, in response to zymosan in rabbit skin were investigated. 2. Oedema formation at skin sites was quantified by the accumulation of intravenously-injected 125I-labelled human serum albumin. 3. Recombinant soluble complement receptor type 1 (sCR1), administered locally in rabbit skin, suppressed oedema formation induced in the RPA reaction and by zymosan. 4. The platelet-activating factor (PAF) antagonists, WEB 2086 and PF10040 administered locally, inhibited oedema formation induced in the RPA reaction and by PAF but not by zymosan. 5. A locally administered leukotriene B4 (LTB4) antagonist, LY-255283, inhibited oedema formation induced by LTB4 but did not inhibit oedema responses to PAF, zymosan or the RPA reaction. 6. The results demonstrate a role for complement in oedema formation in both the RPA reaction and in response to zymosan. An important contribution by PAF is indicated in the RPA reaction but not in response to zymosan whereas no evidence was obtained to suggest a role for LTB4 in either inflammatory response.


The influence of neuronal 5-hydroxytryptamine receptor antagonists on non-cholinergic ganglionic transmission in the guinea-pig enteric excitatory reflex. A partitioned bath made it possible to separate the site of recording of the ascending excitatory reflex of the ileal circular muscle (oral compartment) from the site of reflex induction (caudal compartment), evoked by inflating an intraluminal balloon. In the caudal compartment, blockade of cholinergic ganglionic transmission by hexamethonium (100 microM) and hyoscine (0.3 microM) caused an approximately 65% reduction in the amplitude of reflex contractions, suggesting that the remaining response was mediated by non-cholinergic transmission near the distension site. This non-cholinergic component of ganglionic transmission was insensitive to the action of methiothepin (1 microM), ondansetron (1 microM), tropisetron (1.5 microM), DAU 6285 (1 microM) and renzapride (1 microM), agents that antagonize the action of 5-hydroxytryptamine (5-HT) at neural 5-HT1-like, 5-HT3, 5-HT4 and putative 5-HT1P receptors. These findings suggest that the neural pathways subserving non-cholinergic ganglionic transmission in the ascending excitatory reflex in the guinea-pig ileum do not involve 5-HT as neurotransmitter.


Response to atrial natriuretic peptide, endopeptidase 24.11 inhibitor and C-ANP receptor ligand in the rat. 1. The present studies compared the renal and hypotensive response to (a) exogenous atrial natriuretic peptide (ANP) (99-126), (b) an endopeptidase-24.11 inhibitor (candoxatrilat) and (c) an antagonist of ANP clearance receptors (SC 46542) in conscious rats. 2. Infusion of low-dose-ANP (100 ng kg-1 min-1) produced a gradual increase in urinary sodium and guanosine 3':5'-cyclic monophosphate (cyclic GMP) excretion without significant change in glomerular filtration rate (GFR) or fractional lithium clearance (FeLi). There was a significant fall in blood pressure. 3. Infusion of high-dose ANP (300 ng kg-1 min-1) produced a brisk, 3 fold increase in urinary sodium and cyclic GMP excretion along with a rise in GFR, but had no significant effect on FeLi compared to the control group. The renal response was accompanied by a pronounced fall in blood pressure. 4. Candoxatrilat or SC 46542, alone, had no significant effect on sodium excretion compared to control animals. Both compounds enhanced the natriuretic and cyclic GMP responses to a low-dose ANP infusion (100 ng kg-1 min-1) to levels similar to, or greater than, those observed with the high-dose ANP (300 ng kg-1 min-1). However, unlike high-dose ANP, these renal effects were not accompanied by a significant change in GFR and neither compound potentiated the hypotensive effect of the low-dose ANP infusion. Only candoxatrilat when given with ANP produced a marked rise in FeLi.5. Similarly, combined administration of candoxatrilat and SC 46542 (without exogenous ANP) induced an increase in sodium and cyclic GMP excretion comparable to high-dose ANP but did so without a significant increase in GFR and with a significantly smaller fall in blood pressure. Interestingly, there was no increase in FeLi with the combination of the two compounds, suggesting that the major contribution to sodium excretion came from SC 46542.6. Both candoxatrilat and SC 46542 increased sodium and cyclic GMP excretion in the rat A-V fistula model of heart failure, a model hyporesponsive to infusions of ANP, without significant change in blood pressure.7. These data show that candoxatrilat and SC 46542 do not simply reproduce the effects of an ANP infusion but preferentially enhance the natriuretic response to ANP. Inhibition of E-24.11 may potentiate a tubule action of ANP while the renal mechanism of action of the C-ANP receptor ligand needs further study. Both manipulations are of potential value in the management of heart failure.


Effect of neuropeptide Y on adrenergic and non-adrenergic, non-cholinergic responses in the rat anococcygeus muscle. 1. The effects of neuropeptide Y (NPY) were examined on adrenergic and non-adrenergic, non-cholinergic (NANC) neurotransmission in the rat anococcygeus muscle. 2. NPY (0.1-0.3 microM) greatly potentiated the contractile responses induced by field stimulation. Prazosin (0.1 microM) completely abolished the stimulation-induced responses either in the absence or presence of NPY. 3. NPY (0.1-0.3 microM) enhanced only the contractile responses to low doses of noradrenaline (NA, 0.003-0.01 microM). Responses to tyramine were unaffected by the same concentrations of NPY. 4. In superfused anococcygeus, previously loaded with [3H]-NA, NPY (0.1-0.3 microM) failed to modify the basal, as well as the stimulation-evoked, release of tritium at 2 and 4 Hz. 5. NANC relaxations induced by electrical stimulation were significantly reduced, in a concentration-related manner, by 0.1-0.3 microM NPY. 6. L-NG-nitro-arginine (L-NOARG, 30 microM) enhanced the stimulation (0.25-1 Hz)-induced motor responses. In the presence of L-NOARG (30 microM), NPY (0.1 microM) did not modify the motor responses induced by field stimulation (0.25-0.5 Hz). L-Arginine did not reverse the NPY-induced potentiation of stimulation-induced motor responses. 7. The relaxations of anococcygeus muscle induced by sodium nitroprusside (SNP, 0.01-0.3 microM) were diminished by NPY (0.1-0.3 microM). 8. Our study suggests that NPY, at concentrations devoid of contractile effect, potentiates the motor responses of rat anococcygeus muscle as a consequence, at least in part, of the inhibition of NANC relaxing responses by a different mechanism from L-NOARG.


Albumin inhibits platelet-activating factor (PAF)-induced responses in platelets and macrophages: implications for the biologically active form of PAF. 1. Platelet-activating factor (PAF) binds with high affinity to albumin leading Clay et al. (1990) to suggest that the active form of PAF is the albumin-PAF complex. 2. In the present study the proposal that albumin-bound, rather than monomeric PAF, is the active form of PAF at PAF receptors was critically evaluated by examining the effect of albumin on the potency of PAF in isolated platelets and macrophages. 3. Bovine serum albumin inhibited concentration-dependently PAF-induced responses in platelets and macrophages. The most probable explanation of this finding is that BSA reduced the concentration of free PAF. 4. Thus, we conclude that free PAF, rather than the albumin-PAF complex is the active form. Consequently, local concentrations of albumin will influence profoundly the potency of endogenously released PAF. Moreover, estimates of the affinity of PAF for PAF receptors made in buffers containing BSA, underestimate the true affinity of PAF for its receptors by approximately 3 orders of magnitude.


MDL 26,479: a potential cognition enhancer with benzodiazepine inverse agonist-like properties. 1. The present study investigated biochemical, electrophysiological and behavioural properties of the novel cognition enhancer, MDL 26,479 (5-(3-fluorophenyl)-2,4-dimethyl-3H-1,2,4-triazole-3-thione). 2. The 5-aryl-1,2,4-triazole, MDL 26,479, potently (0.22 +/- 0.05 mg kg-1) inhibited [3H]-flumazenil (Ro15-1788) binding in mouse cortex but was ineffective in vitro at displacing radioligand binding to the GABAA receptor complex. 3. Parenteral administration of MDL 26,479 (1 mg kg-1) or the benzodiazepine (BZD) inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (0.3 mg kg-1) increased cortical ex vivo binding of [3H]-hemicholinium-3 ([3H]-HC-3), a marker for cholinergic activation. This effect of MDL 26,479 was blocked by pretreatment with the antagonist flumazenil (1 mg kg-1). 4. MDL 26,479 (20 microM) and DMCM (1 microM) increased excitation in the hippocampal long-term potentiation (LTP) slice preparation; however, unlike DMCM, the effect of MDL 26,479 was not blocked by flumazenil. 5. In behavioural studies, MDL 26,479 did not exhibit adverse properties characteristic of drugs associated with the GABAA receptor complex. It lacked convulsant, anxiogenic, anxiolytic, or depressant effects. Since MDL 26,479 lacks activity with the BZD receptor in vitro we suggest that it acts via the GABAA receptor complex at another site on this receptor or in an as yet undefined manner or an active metabolite is formed in vivo. 6. Previous work showed that MDL 26,479 enhances learning acquisition in animal models.The present study suggests that at least some of the cognition enhancing properties are due to the enhancement of cortical and hippocampal cholinergic function and LTP.


Reversal by cysteine of the cadmium-induced block of skeletal neuromuscular transmission in vitro. 1. Neuromuscular transmission in isolated nerve-muscle preparations was blocked by exposure to Cd2+ for less than 30 min or more than 2 h. The abilities of cysteine, Ca2+ or 3,4-diaminopyridine (3,4-DAP) to reverse the blockade induced by Cd2+ were studied. 2. On the mouse hemidiaphragm preparation, exposure to Cd2+ (10 microM) for 10 to 20 min induced a blockade which was easily reversed by increasing the extracellular Ca2+ concentration (5-10 mM) or by 3,4-DAP (100 microM). Exposure to Cd2+ (3-10 microM) for over 2 h led to a blockade which was not reversed by Ca2+ (5-15 mM) or 3,4-DAP (100 microM). Cysteine (1 mM) was able to reverse completely the blockade induced by both brief and prolonged exposures to Cd2+. 3. In chick biventer cervicis preparations, Cd2+ (100 microM) decreased the twitch height of indirectly stimulated preparations without affecting responses to exogenously applied acetylcholine, carbachol or KCl. Cysteine (1-3 mM) had no appreciable effect on twitch responses to indirect stimulation or to exogenously applied agonists but fully reversed the blockade induced by Cd2+ (100 microM). 4. In mouse triangularis sterni preparations, Cd2+ (1-30 microM) depressed the evoked quantal release of acetylcholine. Concentrations of Cd2+ which completely blocked endplate potentials (e.p.ps) were without significant effect on miniature endplate potential (m.e.p.p.) amplitude and frequency or time constant of decay. Cysteine (1-10 mM) alone had no effect on e.p.ps or m.e.p.ps, but completely reversed the blockade induced by Cd2+.6. In addition to the competitive blocking action of Cd2+ at the prejunctional Ca2+ channels, long exposure to Cd2+ leads to a blockade that is not competitive. This probably involves binding of Cd2+" at an extracellular thiol site on, or close to, voltage-operated Ca2+' channels.


Non-specific activity of (+/-)-CP-96,345 in models of pain and inflammation. The non-peptide NK1 receptor antagonist, CP-96,345, and its 2R,3R enantiomer CP-96,344, which is not an NK1 receptor antagonist (IC50 > 10 microM), were evaluated for antinociceptive and anti-inflammatory activities in several classical models of pain and inflammation in the rat. Both CP-96,345 and CP-96,344 reduced carrageenin-induced paw oedema and hyperalgesia, and attenuated the second phase of formalin-induced paw licking with equal potency. These results indicate that NK1 antagonism is not responsible for the activity of (+/-)-CP-96,345 in the above animal models.


Characterization of histamine receptor sub-types regulating prostacyclin release from human endothelial cells. 1. The histamine receptor sub-types that are involved in the initiation and maintenance of prostacyclin (PGI2) release from human endothelial cells have been investigated. 2. Endothelial cells cultured from umbilical vein (HUVEC) were incubated with either histamine, the selective H1-receptor agonists, 2-methyl histamine (2-MeHA) or thiazolylethylamine (ThEA), the H1-agonist/H3-antagonist, beta-histidine (beta-His), the selective H2-agonist, dimaprit, the H2-agonist/H3-antagonist, impromidine, the selective H3-agonist, (R)alpha-methylhistamine ((R)alpha-MeHA) and the H3-antagonist, thioperamide. 3. The H1-agonists and the H3-agonist (R)alpha-MeHA induced a concentration (100 nM-1 mM) and time-dependent release of PGI2 as determined by radioimmunoassay for 6-keto-PGF1 alpha, but were less potent than histamine itself. The rank order of potency was the same following 30 min and 24 h incubation, i.e. histamine > ThEA > 2-MeHA >> beta-His > (R)alpha-MeHA. 4. Histamine and 2-MeHA (1 microM-1 mM), ThHEA (10 microM-1 mM) and (R)alpha-MeHA (1 mM), but not beta-His, induced a significantly greater increase in PGI2 release after 24 h incubation than after 30 min incubation (P < 0.05). 5. Neither the selective H2-agonist, dimaprit, nor the H2-agonist/H3-antagonist, impromidine alone induced release of PGI2. 6. The H1-antagonist, mepyramine (10 microM), abolished release of PGI2 induced by histamine, the H1-agonists and (R)alpha-MeHA but the H2-antagonist cimetidine (10 microM) and the H2/H3-antagonist, burimamide (10 microM) did not significantly modulate PGI2 release. 7. Although the H3-agonist (R)alphax-MeHA induced release of PGI2, it failed to modulate PGI2 release in the presence of histamine.8. Low concentrations of the H3-antagonist, thioperamide (100 nM) did not modulate histamine release of PGI2 at all but after 24 h incubation, thioperamide (10-4 M) partially reduced PGI2 release in the presence of histamine.9. These results indicate that PGI2 from HUVEC is initiated and maintained via histamine HI-receptor occupancy. There appears to be no involvement of either H2- or H3-receptors in this particular endothelial cell histaminergic response.


Differential cardiovascular and neuroendocrine effects of epinine and dopamine in conscious pigs before and after adrenoceptor blockade. 1. The effects of epinine or dopamine (both 1-10 micrograms kg-1 min-1) on systemic haemodynamics and plasma concentrations of catecholamines and prolactin were studied in conscious pigs before and after combined non-selective alpha- and beta-adrenoceptor blockade. 2. The plasma concentrations of the two compounds did not differ from each other over the entire dose-range. 3. Epinine increased aortic blood flow (AoBF, 24 +/- 6%), which was due to an increase in heart rate (HR) for doses less than 10 micrograms kg-1 min-1. At 10 micrograms kg-1 min-1, HR decreased slightly (10 +/- 3%, as compared to the value obtained at 5 micrograms kg-1 min-1) and stroke volume increased up to 15% (P < 0.05). Mean arterial pressure (MAP, 99 +/- 3 mmHg at baseline) decreased dose-dependently (14 +/- 2%, P < 0.05) up to the infusion rate of 5 micrograms kg-1 min-1, but increased by 4.0 +/- 1.8 mmHg during infusion of 10 micrograms kg-1 min-1. Systemic vascular resistance (SVR) decreased up to 23 +/- 3% for doses less than 10 micrograms kg-1 min-1, but did not change further during infusion of the highest dose. LVdP/dtmax increased during the two highest infusion rates up to 22 +/- 6% (P < 0.05). After the infusion was stopped there was an abrupt increase in HR (18 +/- 4%, P < 0.05) and a further decrease in SVR before all parameters returned to baseline.4. Dopamine caused increases in AoBF (27 +/- 3%) similar to epinine, the only difference being that HR continued to increase (32 +/- 5%) and MAP (13 +/- 3%) and SVR continued to decrease (31 +/- 3%) over the entire dose-range. The increase in LVdP/dt,,,, at the highest dose (48 +/- 4%, P <0.05) was more pronounced than with epinine.5. Adrenoceptor blockade inhibited all epinine-induced changes, but did not affect the dopamineinduced changes in AoBF, SVR and MAP, but attenuated the increases in HR and LVdP/dtmax.6. Noradrenaline (NA) and adrenaline (Ad) concentrations did not change during infusion of epinine or dopamine, but NA increased by 50% within 2.5 min after stopping the infusion of epinine. After adrenoceptor blockade NA and Ad concentrations did not change during infusion of dopamine, which contrasted with a decrease of 55 +/- 5% (P<0.05) in NA during infusion of epinine.7. Prolactin concentrations decreased gradually from 480 +/- 40 pg ml-' to 270 +/- 50 pg ml1' (P<0.05) during infusion of epinine, but did not change significantly during dopamine infusion.8. The differential effects of epinine and dopamine on MAP, SVR, plasma NA (before and after adrenoceptor blockade) and prolactin, leads us to conclude that in conscious pigs, epinine is a more potent a, P2 and D2-receptor agonist, but a weaker D,-receptor agonist than dopamine.