Similar prevalence of enteroviral genome within the myocardium from patients with idiopathic dilated cardiomyopathy and controls by the polymerase chain reaction. To assess the prevalence and significance of enteroviral genome within myocardial biopsy specimens taken from patients with idiopathic dilated cardiomyopathy and from controls. Prospective evaluation of myocardial tissue for the presence of an enteroviral genome by the polymerase chain reaction. A tertiary referral centre for patients with idiopathic dilated cardiomyopathy. Tissue for the study came from 50 consecutive patients with dilated cardiomyopathy, 41 with other forms of heart disease and 34 from coroners' necropsy cases. Enteroviral genome was detected in 6/50 (12%) patients with dilated cardiomyopathy and 13/75 (17%) of the controls (not significant). No differences were seen between dilated cardiomyopathy patients with or without myocardial enteroviral genome in respect of age; duration of symptoms; proportion of patients with a premorbid acute viral illness, excess alcohol consumption, or hypertension; New York Heart Association functional class; measures of left ventricular function; or endomyocardial histology. Within the control group enteroviral genome was detected in 3/15 (20%) patients with ischaemic heart disease, 2/19 (10.5%) with valvar heart disease, 1/5 (20%) with specific heart muscle disease, 0/2 (0%) with congenital heart disease, and 7/34 (20.6%) cases of sudden death. During 2-52 month follow up (mean 22) 15/44 (34%) patients without myocardial enteroviral genome and 2/6 (33%) with myocardial enteroviral genome died suddenly or required orthotopic heart transplantation for progressive heart failure. These findings do not support the hypothesis that persistent enteroviral infection is of pathogenic or prognostic importance in dilated cardiomyopathy but they are consistent with enterovirus being a common environmental pathogen.


Evaluation of spin resonance spectroscopy of red blood cell membranes to detect malignant hyperthermia susceptibility. We have evaluated a spin labelled electron spin resonance technique to identify malignant hyperthermia susceptible (MHS) patients. We studied 19 patients, 10 MHS and nine MHN (normal), using the standard European procedure. We were unable to obtain any evidence that this technique could be used to diagnose MH susceptibility. Furthermore, there was no significant difference in the fluidity of the red blood cell membranes between the two groups, which would have been indicative of a generalized membrane abnormality in MH.


Interaction between halothane and mu, delta and kappa opioid agonists on the isolated right atria of the rat. We have examined the interaction between halothane and specific opioid agonists at mu (morphine and [D-ala2 N-mephe4, gly-ol5]-enkephalin (DAGO)), delta ([D-pen2,5]-enkephalin (DPDPE)) and kappa (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrroliknyl)cyclohexyl]- bencetamide methane sulphonate (U-50,488H)) receptors on the isolated right atria of the rat. All the opioid agonists tested decreased atrial rate. The maximal effects obtained with U-50,488H (75 (SE 3.3)%) were significantly (P < 0.001) greater than those obtained with morphine (12 (2.7)%), DAGO (8 (0.6)%) or DPDPE (11 (1.8)%). Halothane 1.5 v/v% did not modify the inhibitory effects induced by morphine, DAGO or DPDPE. However, U-50,488H had a potentiating effect in the presence of halothane 1.5 v/v% (P < 0.001). Naloxone 5 x 10(-7) and 1 x 10(-6) mol litre-1 antagonized the inhibitory effects of U-50,488H in the presence of halothane. We conclude that halothane increased the potency of a kappa agonist on isolated right atria and suggest that this effect was mediated by opioid receptors.


Pharmacokinetics of mivacurium in normal patients and in those with hepatic or renal failure. We have determined the pharmacokinetics and duration of action of a bolus dose of mivacurium (0.15 mg kg-1) during isoflurane and nitrous oxide anaesthesia in nine patients with normal renal and liver function, nine patients undergoing cadaveric kidney transplantation and nine patients undergoing cadaveric liver transplantation. Total plasma concentrations of mivacurium were measured for 2.5 h after administration using a high-pressure liquid chromatographic assay. Plasma concentration vs time data for what were presumed to be the two active mivacurium isomers were analysed by a non-compartmental method based on statistical moments. Neuromuscular block was assessed by measuring the electromyographic evoked response of the adductor pollicis muscle to train-of-four stimulation of the ulnar nerve. The mean time to recovery of 25% neuromuscular transmission, T25, was greater in the patients with liver failure (57.2 min) than in control patients (18.7 min). The volume of distribution at steady rate (Vdss) was comparable in the three groups. Patients with impaired liver function had significantly longer mean residence time and smaller plasma clearance than did patients with renal failure or control patients. There were significant negative correlations between plasma cholinesterase activity and both T25 (r = 0.79) and mean residence time (r = 0.62).


[Malignant fibrous histiocytoma of the pancreas]. The authors report a case of pancreatic malignant fibrous histiocytoma (approximately 22 cm). Clinical and instrumental approach for preoperative diagnosis is discussed and the anatomopathological features of the lesion, which is rarely localized in the pancreatic gland, are stressed. Surgical treatment is outlined as well.


Two roles for transforming growth factor beta 1 in colon enterocytic cell differentiation. The role of transforming growth factor beta 1 (TGF-beta 1) in enterocytic differentiation was examined by treating two undifferentiated HT29 colon carcinoma sublines, U4 and U9, with hexamethylene bisacetamide to up-regulate their level of TGF-beta 1 mRNA expression. Although both lines after treatment secreted approximately equal levels of biologically active TGF-beta 1, only U4H cells were found to undergo enterocytic differentiation when cultured postconfluence on collagen I-coated transwells, forming polarized monolayer cells with an apical brush border, whereas U9H cells remained multilayered and undifferentiated. Enterocytic U4H cells exhibited four times as much cell surface expression of the collagen I-binding protein alpha 2-integrin, twice as much of the accessory collagen-binding protein carcinoembryonic antigen, and almost twice as much binding to collagen I films as undifferentiated U9H cells. TGF-beta 1 treatment doubled U4 cell collagen I binding, increased expression of alpha 2-integrin 4-fold, but increased carcinoembryonic antigen expression only marginally. U4H cells displayed cell cycle regulation by arresting reversibly at a restriction point in G1 when placed in the postconfluent culture conditions which initiated enterocytic differentiation. In contrast, undifferentiated U9H cells exhibited no restriction point but arrested throughout G1. TGF-beta 1 blocked synchronized U4H cells in G1, whereas it stimulated the growth of U9H cells. Thus, TGF-beta 1 has two roles in enterocytic differentiation: to increase levels of collagen I adhesion proteins and to block enterocytic cells in G1 so that they can differentiate.


Retinoic acid induces secretion of latent transforming growth factor beta 1 and beta 2 in normal and human papillomavirus type 16-immortalized human keratinocytes. Similar cellular responses are elicited by retinoic acid (RA) and transforming growth factor beta (TGF-beta). We investigated the ability of RA to modulate the production of TGF-beta in normal human keratinocytes (HKc) and HKc lines immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16). RA treatment of both normal HKc and HKc/HPV16 resulted in a 2-3-fold induction in secreted levels of latent TGF-beta. The induction in TGF-beta secretion by RA was dose dependent, with significant increases observed with RA concentrations as low as 1-10 nM, and time dependent, with maximal induction occurring about 3 days after initiation of RA exposure. In addition, RA induced intracellular levels of TGF-beta almost 5-fold. Sandwich enzyme-linked immunosorbent assays were used to specifically quantify TGF-beta 1 and TGF-beta 2 secreted by normal HKc and HKc/HPV16 cultured in the absence or presence of RA. RA increased the secreted levels of latent TGF-beta 1 and TGF-beta 2 an average of 2- and 5-fold, respectively, with no major differences in the fold induction between normal HKc and HKc/HPV16. Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced specific transcripts for TGF-beta 1 and TGF-beta 2 about 3- and 50-fold, respectively. RA treatment of HKc had no significant effect on the binding affinity of TGF-beta for its receptors or receptor number. Normal HKc and HKc/HPV16 displayed similar dose-dependent inhibition of proliferation by TGF-beta 1. These studies indicate that RA may regulate growth control in both normal HKc and HKc/HPV16 by enhancing TGF-beta 1 and TGF-beta 2 production, which, after activation at the cell surface, could inhibit cellular proliferation in an autocrine and/or paracrine manner.


[The circulation of the hepatitis A and B viruses in the Somali population]. We report the results of a study carried out to evaluate the extent of hepatitis A virus (HAV) and hepatitis B virus (HBV) circulation in Somalia. Serum samples were collected from 593 subjects (age range 0-83 years) and tested for anti-hepatitis A (HAV) and anti-HAV IgM. Serum samples taken from 1272 individuals (age range 0-83 years) were tested for HBsAg, anti-HBsAg, anti-HBcAg, HBeAg and anti-HBeAg. We confirmed a very high rate of HAV exposure (about 90% of the subjects tested had circulating anti-HAV) as is typical of fecal-orally transmitted infectious agents. The age-specific anti-HAV IgM prevalence suggests that HAV infection is acquired very early in life. Our data also indicate a high rate of HBsAg carriers (range: 10.5%-27.4%) in the Somalian population. When all markers are considered, 60% of the adult population showed evidence of HBV exposure. HBV spreads very subtly: in fact, it is generally transmitted via non-overtly percutaneous routes. In Somalia, hepatitis A virus infection is highly endemic and occurs very early in life. Hepatitis B virus infection is also widespread in this country.


Expression of the HPV16 E7 gene generates proliferation in stratified squamous cell cultures which is independent of endogenous p53 levels. Monolayer cultures of human foreskin and ectocervical epithelial cells were infected with retroviral vectors expressing HPV16 oncogenes, selected for G418 resistance, and cultured organotypically so that they reformed the fully differentiated, stratified squamous tissues from which they were originally derived. Expression of HPV16 E7 prevented cell cycle withdrawal in the suprabasal layers of these stratified cultures but had no effect on terminal differentiation. Cultures expressing E7 alone and those coexpressing E6 and E7 were identical in terms of suprabasal proliferation and terminal differentiation, but they differed in expression of the endogenous tumor suppressor protein p53. Immunohistochemically detectable p53 protein localized to the proliferative compartment in normal and E7-containing cultures but was undetectable in those cultures which coexpressed E6 and E7. This result suggests that E7-induced suprabasal proliferation is independent of the steady-state level of p53.


Hepatocellular carcinoma and sex hormones. The liver is morphologically and functionally modulated by sex hormones. Long-term use of oral contraceptives and androgenic steroids can induce benign and malignant hepatocellular tumors. Hepatocellular carcinoma (HCC) is more prevalent in men than in women. The role of sex hormones and their receptors in the development of HCC was reviewed. Some HCCs may be androgen dependent but others may be estrogen or even both dependent. Further studies are mandatory in order to utilize such characteristics of HCC for an effective prophylaxis and therapy of this tumor.


[Lymphokine-activated killer cell adoptive immunotherapy for cancer treatment and its significance]. New culture system, CDCS-T1, was developed for clinical conduction of lymphokine-activated killer (LAK) cell adoptive immunotherapy (AIT). Advanced or recurrent cancer patients of digestive tract were treated with AIT with LAK cells generated by CDCS-T1 in combination with plasma exchange. Partial responses were shown in 10 to 20% of patients treated. Long survival was found in some responders, indicating the significance of LAK therapy for cancer treatment. AIT with LAK cell transfer was also conducted in patients with esophageal cancer as postoperative adjuvant therapy. Better restoration of postoperative depression of immunological parameters was found in patients with postoperative LAK cell transfer. It is suggested that postoperative LAK cell transfer is a good candidate for adjuvant immunotherapy for cancer treatment.


[Suppression of myeloperoxidase gene expression by gamma-interferon]. The gene expression of myeloperoxidase (MPO) in HL60 cells, as tested by Northern blot analysis with a MPO gene probe, was markedly suppressed by treatment with gamma interferon (IFN-gamma)(200U/ml) for 9hr, whereas cytochemically detected MPO activity, cell surface antigen expression, and cell morphology remained unchanged even at 48hr after the treatment. IFN-gamma of 50U/ml was sufficient for the suppression at 24hr. When the HL60 cells treated with IFN-gamma (200U/ml) for 24hr were cultured in the absence of IFN-gamma for another 24hr, the transcript of MPO gene reverted to a level comparable to that of the HL60 cells cultured thoroughly in the absence of IFN-gamma, indicating the reversibility of the suppression. The suppression of MPO expression at RNA level, possibly independent of differentiation, is one of the biological activities exerted by IFN-gamma, which has not been previously reported.


Kinetics of amide proton exchange in parvalbumin studied by 1H 2-D NMR. A comparison of the calcium and magnesium loaded forms. The amide proton exchange rates have been measured for the pike parvalbumin loaded either with calcium (PaCa2) or with magnesium (PaMg2) by using 2-D total correlation spectroscopy experiments. The differences in the exchange rates observed between these two species were unexpected when compared with the small conformational changes induced in parvalbumin by the Ca/Mg exchange. With the calcium-loaded protein (PaCa2), a significant difference was observed for the amide proton exchange rates of residues located in the N-terminal domain AB in contrast to the slower exchange rates that were observed in the CD and EF domains. Such a difference does not exist for PaMg2, where faster exchange rates are observed over all the sequence. Since amide proton exchange rates are the signature of the solvent's accessibility in proteins, we interpreted our results in terms of difference of the equilibria between 'closed-states' and 'opened-states' for individual amide protons of the protein when calcium was replaced by magnesium. The CD and EF domains, and to a lesser extent the AB domain, would be more rigid when the protein was loaded with calcium ions. For the magnesium-loaded parvalbumin (PaMg2) the faster exchange rates we observed could be rationalized by a more flexible structure than in the case of the PaCa2.


The seven-stranded beta-barrel structure of apo-neocarzinostatin as compared to the immunoglobulin domain. The three-dimensional structure of apo-NCS, as revealed by proton NMR, is based on an antiparallel seven-stranded beta-barrel. This fold is frequently encountered in protein structures, especially for immunoglobulin domains. The strands forming the barrel are joined by flexible loops of which three are implicated in the ligand binding site of these proteins. In this paper a preliminary comparison is given with respect to the static and dynamic properties of both the constant beta-barrel and the active loops for apo-NCS and the variable VH domain of an immunoglobulin Fab' fragment.


Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies. The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.


Two-dimensional 1H NMR spectroscopy of normal and pathological human plasma: complete water suppression and further assignment of resonances. Two-dimensional J-resolved and correlated 1H NMR spectra with complete water suppression have been obtained to further characterize a metabolic pattern for normal and pathological human plasma samples. 1H COSY spectra have been recorded on plasma from 12 patients with cancer in order to check for the possible presence of fucose. Our results show that there is no evidence for the presence of fucosylated lipids in the plasma of these patients.


[Characteristics of chloramine complexes of carnosine with hypochlorite anion]. Carnosine interaction with CIO results in the formation of a stable chloramine complex. The binding of the whole bulk of hypochlorite to carnosine is completed within one minute of incubation. During subsequent 2-hour incubation no more than 15% of the chloramine complex is destroyed; this property of carnosine makes it similar to taurine. Unlike histidine and beta-alanine, glutathione rapidly interacts with hypochlorite. However, in contrast with these compounds and carnosine, glutathione does not form stable chloramine complexes with CIO. The putative role of myeloperoxidase in the development of senile human lens opacities is discussed.


Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture. Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.


[The effect of carnosine and trolox on cellular chemiluminescence of leukocytes]. The effects of hydrophilic antioxidant carnosine, trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid), and superoxide dismutase on the myeloperoxidase activity of leukocytes, superoxide anion and active oxygen species generation have been studied. Physiological concentrations of carnosine have been shown to decrease the ability of human leukocytes to produce chemiluminescence as a result of myeloperoxidase activation. However, the chemiluminescence induced by the generation of the superoxide or its derivatives is unaffected by this process. Trolox does not inhibit the induction of superoxide-dependent chemiluminescence of leukocytes either.


[The effect of carnosine on the activity of Na,K,ATPase: prospective uses in clinical cardiology]. The effects of carnosine on erythrocyte membrane Na,K-ATPase and isolated enzyme in vitro as well as on membrane Na,K-ATPase activity and lipid peroxidation (LPO) in chronic heart failure (CHF) and acute myocardial infarction (AMI) have been studied. CHF and AMI have been shown to be associated with significant inhibition of the erythrocyte membrane Na,K-ATPase activity and LPO activation. Marked activation of erythrocyte membrane Na,K-ATPase by carnosine in comparison with the isolated enzyme has been established. The ability of carnosine to induce Na,K-ATPase activation and prevent membrane depolarization indicates that the dipeptide may be a useful tool in the pathogenetic therapy of CFH and AMI.


[Direct measurement of the interaction of carnosine and its analogs with free radicals]. An ESR study of interactions of carnosine and its derivatives with free radicals has been carried out. In model systems the spin trap OH. radical adduct generation has been shown to decrease significantly in the presence of carnosine in a pronounced concentration-dependent manner. A comparative study of effects of some other histidine-containing dipeptides on this process has revealed a similarity in anserine, homocarnosine, and acetylcarnosine actions.


A 13C NMR study on fluxes into the TCA cycle of neuronal and glial tumor cell lines and primary cells. Two tumor cell lines (C6 glioma and N1E-115 neuroblastoma), primary glia and primary neurons (from rat) were incubated with 2-13C-pyruvate and 3-13C-pyruvate in culture dishes. 13C NMR spectra of the cell extracts were used to determine the ratio of pyruvate carboxylase to pyruvate dehydrogenase activity. Pyruvate carboxylase activity was found higher in primary glia cells than in neurons. Glial cells synthesized more amino acids, ie, their TCA cycle was used to a larger extent for biosynthesis than is the case of neurons, where it is preferentially used for the energy metabolism.


[The use of carnosine in medical practice. Priorities: past and future]. The history of the discovery of curative effects of carnosine and its perspective applications in the clinical practice are reviewed. The molecular mechanisms of carnosine interactions with free oxygen radicals (hypochlorite anion, in particular) are considered.


Energetic metabolism of glucose, mannose and galactose in glucose-starved rat insulinoma cells anchored on microcarrier beads. A phosphorus-31 NMR study. Insulin-secreting cells (RINm5F) have successfully been grown on a large scale on poly-L-lysine coated-polystyrene microcarriers, providing a high cell number in a restricted volume under conditions that respect the metabolic integrity of these anchorage-dependent cells. The energetic metabolism of the perfused cells has been followed non-invasively by phosphorus-31 nuclear magnetic resonance spectroscopy. Glucose starvation induced a rapid decrease in nucleoside triphosphates (mainly ATP) pools, correlated with an increase in Pi level. The initial ATP level was rapidly recovered when the cells were refed with glucose or with mannose, but not with galactose, even after 2 h of perfusion. These differential effects of hexoses on energetic metabolism might be related to their various insulin-release actions on tumor islet cells.


[Genomic instability and AIDS].

[Genomic instability and AIDS]. The biochemical mechanisms underlying blood lymphoid cell genome destabilization in patients with HIV infection have been analyzed. Lymphocytes from HIV patients are characterized by increasing intensity of free radical oxidation together with activation of the xanthine oxidase D-form conversion into the O-form, enhanced activity of UV-endonuclease, and intensification of prooxidant-induced proteolysis. These changes increasing with the progress of the disease with a maximum at the AIDS stage form a metabolic basis for labilization of the lymph cell genome. The degree of biochemical manifestations of genome instability (levels of chromatin degradation products and intensity of formation of one-filament nicks of DNA) increase in the dynamics of HIV-infection. The data obtained are discussed in terms of the author's conception on the origin of AIDS from retroposons (retrotransposons?). A hypothesis is postulated on accumulation of autonomous genetic information on the basis of genome labilization under the influence of genotoxic factors. Clinico-biochemical data on the appearance of HIV proteins (p17, p24) in the blood of patients (previously negative for all HIV markers) in the presence of transfusions of HIV-negative blood and UV-irradiation of the autoblood are also discussed from this standpoint.


Secretion of thioredoxin after in vitro activation of human B cells. The redox-active enzyme thioredoxin (Trx) is secreted by various virus-transformed cell lines of B- and T-cell origin and has been considered to play an autoregulatory role as a cofactor during cellular growth processes. We show in this paper that exposure of B lymphocytes from normal, healthy donors and B cells from B-type chronic lymphocytic leukemia (B-CLL) to Staphylococcus aureus Cowan I (SAC) induced expression of Trx mRNA. By combining SAC, or the phorbol ester TPA, with IL-2 and the conditioned medium of a T-cell hybridoma (BSF-MP6), we could strongly enhance the Trx expression. After [35S]methionine labeling of stimulated B-CLL cells in vitro, Trx was immunoprecipitated both from cell extracts and from the medium with antibodies against human placenta Trx. Secretion of newly synthesized Trx was also confirmed by a quantitative radioimmunoassay for human Trx. During 24 h cultivation experiments, treatment with SAC induced a 5-fold increase of the Trx content of normal B lymphocytes as well as in B-CLL cells. Approximately two-thirds of the total amount of the enzyme was released into the medium.


Epstein-Barr virus BCRF1 gene product (viral interleukin 10) inhibits superoxide anion production by human monocytes. Due to its similar biological activities to interleukin 10 (IL-10), Epstein-Barr virus (EBV) BCRF1 gene product (viral IL-10: vIL-10) has recently been recognized as an analogue of authentic IL-10. Preincubation of human monocytes with vIL-10, like human IL-10, induced smaller amounts of interferon-gamma (IFN-gamma) mRNA in activated human peripheral blood mononuclear cells (PBMNCs) than nonpreincubation, indicating that vIL-10 acts principally on monocytes. Since the activation of monocytes and their generation of oxidative products are regulated by various cytokines, we examined the effects of vIL-10 on superoxide anion (O2-) production by human PBMNCs and monocytes. Not only PBMNCs but also monocytes preincubated with vIL-10 showed a smaller production of O2-. Inhibition was achieved in a dose-dependent fashion and increased gradually after incubation with vIL-10. Additions of IFN-gamma, macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), which prime monocyte activation and induce O2- production, were also affected by the reciprocal effect of vIL-10. Thus, vIL-10 production by EBV-infected cells may be involved in the development of EBV-related disorders.


Adrenal steroid receptor activation in rat brain and pituitary following dexamethasone: implications for the dexamethasone suppression test. The dexamethasone suppression test (DST) has been used extensively to evaluate feedback inhibition of the hypothalamic-pituitary-adrenal (HPA) axis by adrenal steroids. Nevertheless, it remains unclear at what level of the HPA axis and through which adrenal steroid receptor subtype dexamethasone exerts its inhibitory effect. Because adrenal steroid receptor activation is an important prerequisite for dexamethasone to affect cellular function, HPA axis tissues that exhibit evidence of receptor activation following dexamethasone administration are likely site(s) of action for this synthetic hormone to inhibit HPA axis activity. Therefore, type-I and type-II adrenal steroid receptor activation was assessed in the pituitary, hypothalamus, and hippocampus of intact and adrenalectomized rats after overnight exposure to various oral doses of dexamethasone. Results with dexamethasone were compared to similar studies using corticosterone, the endogenous glucocorticoid of the rat. All dexamethasone doses led to significant type-II receptor activation in the pituitary, whereas only an exceedingly high dexamethasone dose activated type-II receptors in the hippocampus and hypothalamus. Dexamethasone had little effect on type I receptors in any tissue at any dose. In contrast, corticosterone significantly activated type-I receptors in all tissues, whereas it activated type-II receptors in the brain and not the pituitary at physiological concentrations. Because dexamethasone activated pituitary type-II receptors at blood concentrations that did not activate type-II receptors in the brain, these results suggest that the DST in humans may primarily be a measure of type-II adrenal steroid receptor feedback inhibition at the level of the pituitary.


Effects of alprazolam on pituitary-adrenal and catecholaminergic responses to metabolic stress in humans. Concurrent effects of benzodiazepines on stress-induced activation of the three classical "stress" systems: pituitary-adrenal, adrenomedullary, and sympathoneural systems have not been extensively investigated in humans. In the present study, the effects of alprazolam (1.5 mg) on plasma levels of adrenocorticotropin hormone (ACTH), epinephrine, norepinephrine, dihydroxyphenylglycol (DHPG, the intraneuronal metabolite of norepinephrine), and mood states were examined in 10 healthy volunteers undergoing glucoprivic stress. Glucoprivic stress was induced by intravenous administration of the glucose analog, 2-deoxyglucose (2DG), at a dose (50 mg/kg) that impairs cellular glucose metabolism and produces a state comparable to hypoglycemia. Alprazolam and 2DG were administered in a double-blind, placebo-controlled manner. 2DG produced robust elevations in plasma ACTH and epinephrine levels, modest elevations in plasma norepinephrine levels, and decreases in plasma DHPG levels. Alprazolam significantly attenuated the 2DG-induced increases in plasma ACTH and epinephrine, but did not significantly effect plasma norepinephrine and DHPG. These data suggest that benzodiazepines attenuate metabolic stress-induced activation of the pituitary-adrenal and adrenomedullary systems but do not effect 2DG-related effects on peripheral sympathoneural function. The possible mechanisms involved are discussed.


Benzodiazepine binding sites and their modulators in hippocampus of violent suicide victims. Benzodiazepine binding sites were studied by autoradiography in several hippocampic layers in brains of drug-free violent suicide victims (hanging) and matched controls. Kd was increased in suicides, and when brain sections from control subjects were incubated in the bath fluid that had previously served to incubate sections from suicides, Kd was increased in the same way. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA and amino acid concentrations.


Thermally induced chain exchange of frog alpha beta-tropomyosin. The thermally induced unfolding of the alpha-helix of Rana esculenta alpha alpha, alpha beta and beta beta tropomyosin and two tryptic fragments approximately corresponding to the N- and C-terminal halves of alpha beta have been investigated by use of optical rotation, circular dichroism and UV difference spectroscopy. Reversible unfolding transitions of alpha alpha and beta beta occur around 49 degrees C and 32 degrees C, respectively. The helix unfolding of alpha beta shows two major transitions at 36 degrees C and 48 degrees C, with only the latter being reversible. The major unfolding transitions of each of the N- and C-terminal alpha beta peptides roughly correspond to the low and high temperature transitions of intact alpha beta, respectively. This suggests that the unfolding of alpha beta could be due to unfolding of two independent domains in alpha beta. UV difference data, crosslinking and chromatography results show, however, that the unfolding of alpha beta at 36 degrees C is due to chain exchange with the formation of alpha alpha homodimers and largely unfolded beta monomers, and that the transition at 48 degrees C is due to unfolding of alpha alpha dimers.


Homochirality and long-range transfer in biological systems. It has been shown that the chiral purity of biomacromolecules has important biological significance not only from the standpoint of lock-and-key stereocomplementarity, but also as a basis for long-range communication in biosystems. An explicit demonstration is given for the case of proton transfer along the hydrogen-bonded chain that is formed by amino acids containing OH groups. It is found that the replacement of the L-amino acid residue by the D-isomer in a peptide chain suppresses proton transport through the hydrogen bond network.


Structured Biological Modelling: a method for the analysis and simulation of biological systems applied to oscillatory intracellular calcium waves. In biology signal and information processing networks are widely known. Due to their inherent complexity and non-linear dynamics the time evolution of these systems can not be predicted by simple plausibility arguments. Fortunately, the power of modern computers allows the simulation of complex biological models. Therefore the problem becomes reduced to the question of how to develop a consistent mathematical model which comprises the essentials of the real biological system. As an interface between the phenomenological description and a computer simulation of the system the proposed method of Structured Biological Modelling (SBM) uses top-down levelled dataflow diagrams. They serve as a powerful tool for the analysis and the mathematical description of the system in terms of a stochastic formulation. The stochastic treatment, regarding the time evolution of the system as a stochastic process governed by a master equation, circumvents most difficulties arising from high dimensional and non-linear systems. As an application of SBM we develop a stochastic computer model of intracellular oscillatory Ca2+ waves in non-excitable cells. As demonstrated on this example, SBM can be used for the design of computer experiments which under certain conditions can be used as cheap and harmless counterparts to the usual time-consuming biological experiments.


Ca2+ and partly GTP gamma S-dependent particulate phospholipase C hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is inhibited by diacyl(acyl-acetyl) glycerols. The activity of a phosphodiesterase of the phospholipase C (PLC) type and factors influencing its activity were studied in ascites tumor cells. The enzyme confined to the 12,000 x g particulate fraction hydrolyses inositol phospholipids, with preference for phosphatidylinositol 4-phosphate (PtdIns(4)P) over phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), exhibiting maximum values of 61 and 15 nmol/min per mg protein, respectively, at a pH optimum of 5.5. The phosphodiesterase, which is strongly Ca2+ dependent with optimal free Ca2+ concentrations between 20 and 100 nM for both substrates, is almost completely inhibited (93-95%) in the presence of 2 mM EGTA. Only the PLC acting on PtdIns(4,5)P2 is significantly activated in the presence of 6-60 microM GTP gamma S. The low extent of enzymatic activity in the presence of 5 mM MgCl2 or chelating agents is suggestive of inositolphosphatase activity which is supported by the determination of small amounts of myo-inositol during HPLC analyses. Both dioleoylglycerol (DAG) and the membrane-permeable 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibit PLC activity, exhibiting IC50 values of 5 microM with PtdIns(4)P and approx. 10 microM with PtdIns(4,5)P2 as substrate and maximum inhibition up to 60% (DAG) and 80% (OAG). These data are indicative of a mechanism of direct negative feedback regulation of the enzyme by diglycerides which may explain the observed long-term effects of OAG on PLC activity in cell culture experiments.


Chagasic IgG stimulates phosphoinositide hydrolysis via neurotransmitter receptor activation: role of calcium. Induction of polyphosphoinositide hydrolysis in cardiac tissue by IgG from chagasic mice was assayed. BALB/c mice auricles were labelled with myo-[3H]inositol precursor and inositol phosphate production in the presence or absence of chagasic IgG and the corresponding F(ab')2 was measured. Both chagasic IgG and F(ab')2 but not the normal forms specifically increased phosphoinositide turnover. This increment was blocked by muscarinic cholinergic antagonists and to an even greater extent by the phospholipase C inhibitor NCDC. Moreover, calcium channel blocking agents such as diltiazem, verapamil and D-600 also exerted an inhibitory action. A muscarinic cholinergic agonist, carbachol, and the ionophore A-23187, mimicked the action of the chagasic IgG upon phosphoinositide turnover. It is concluded that murine chagasic IgG and its F(ab')2 fragments result in stimulation of phospholipase C-mediated phosphoinositide hydrolysis through the interaction with muscarinic cholinergic receptors requiring the cytosolic calcium concentration to be raised.


Increased platelet-activating factor (PAF) concentrations in hearts and lungs of Pichinde virus-infected guinea pigs. Platelet-activating factor (PAF) has been implicated as a cause of cardiopulmonary disturbances in certain diseases. In the present study, concentrations of PAF in hearts, lungs, whole blood, and other organs of control and Pichinde virus-infected guinea pigs on post-inoculation days (PID) 10 and 14 were measured by radioimmunoassay. Results were further confirmed by bioassay after separation and purification with thin-layer chromatography. PAF concentration in the hearts and lungs of virus-infected animals increased significantly on PID 10 and 14, as compared with control levels. PAF level in the blood of infected guinea pigs also significantly increased on PID 14. There was little change of PAF levels in liver and kidney after viral inoculation. Increased PAF concentrations in both hearts and lungs of infected strain 13 guinea pigs suggest that this lipid mediator may play an important role in the development of cardiopulmonary disturbances.


Pregnancy-associated remodeling of rabbit endometrial platelet-activating factor receptors. [3H]PAF binding parameters (affinity constants and binding capacities) were estimated for endometrial membranes obtained on days 3 (when the morulae first enter the gravid uterus), 4, 5 and on day 6, about 22 h before the blastocyst becomes irremovably attached to the endometrium. The two-site receptor system which characterized the binding on a large proportion of purified membranes from days 3 (60%) and 6 (75%) had the following parameters: day 3, Kd1 (nM) 0.15 +/- 0.04, Bmax1 (pmol/mg protein) 0.04 +/- 0.01; Kd2 (nM) 17.60 +/- 6.40, Bmax2 (pmol/mg protein) 2.92 +/- 0.91 (n = 6), and day 6, Kd1 (nM) 0.33 +/- 0.06, Bmax1 (pmol/mg protein) 0.11 +/- 0.02; Kd2 (nM) 7.42 +/- 1.02, Bmax2 (pmol/mg protein) 1.75 +/- 0.31 (n = 3). The remaining membranes, including all preparations of days 4 and 5, exhibited only one class of binding sites which were unlike the putative type 1 PAF receptor. The most significant observations were the apparent pregnancy-associated changes in the parameters of the PAF binding sites. However, regardless of the type of PAF binding exhibited, all the binding sites bound GTP. [3H]PAF dissociation from bound complexes was biphasic at 25 degrees C. The rate constants were k-1, 0.27 +/- 0.09 min-1 and k-2, 3.45 +/- 1.19 x 10(-3) min-1, for the rapid and slow dissociating components, respectively. In view of the inability to resolve the experimental data from days 4 and 5 into multiple sites, and of the occurrence of receptor interconversions on days 3 and 6, it is postulated that there may be cell membrane macromolecular reorganization/remodeling of proteins and cytoskeleton as a result of the arrival of blastocyst in the uterus and/or the dynamic interplay of local uterine PAF and the hormonal milieux during the peri-implantation period of pregnancy.


Effect of platelet agonists on the binding of platelet-activating factor to human platelets. The binding of PAF in vitro to its receptors on platelets gradually becomes irreversible. This property has been applied to the measurement of PAF in circulating blood, the concept being that in vivo contact with endogenous PAF diminishes subsequent in vitro binding of 3H-labeled PAF. Here we show that, when platelets are stimulated with thrombin, the subsequent binding of [3H]PAF is greatly reduced, probably due to proteolytic damage of the PAF receptors. In addition, [3H]PAF binding is decreased, when the cell's energy status is low, which is seen during thrombin-induced secretion. Thus, the detection of PAF in circulating blood based on the binding to PAF receptors on platelets is severely hampered when platelets make contact with thrombin.


[ACTH inhibits the development of primary decompensation of the systemic and portal circulation in acute blood loss in rats]. Using the method of contact luminescent biomicroscopy of the liver and intestine coupled with the measurement of systemic blood pressure by micromanometer and ultrasonic registration of blood flow velocity in portal vein and hepatic artery it has been established that in rats with acute decompensatory hemorrhage fragments of ACTH (1-24) and (4-10) improve the state of portal macro- and microcirculation and increase the life span 2-3-fold. ACTH does not influence the dynamics of acute compensatory hemorrhage and the development of the posthemorrhagic microcirculatory disturbances (local microstases, microthromboses, erythrocyte aggregation).


[Alpha 1-adrenergic receptors in the liver parenchyma of children: the changes in cirrhosis]. In the present study alpha 1-adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of prehepatic portal hypertension children without parenchymal affection (control group, n = 7) and the resection of children in parenchymal affection (group of cirrhosis, n = 8). It has been shown, that the binding of alpha 1-adrenergic antagonist 3H-prasozin (3H-PRZ) in liver parenchyma membranes of both control and cirrhosis groups is saturable and shows a high affinity. The Scatchard analysis of the binding data indicated that the binding site is characterized by Kd and Bmax of 0.6 +/- 0.12 nM, 92.8 +/- 8.0 fmol/mg, respectively, for the control group; and 1.5 +/- 0.4 nM, 254.1 +/- 28.4 fmol/mg, respectively, for the group of cirrhosis; (mean +/- SEM). It has been found that the number of binding sites of 3H-PRZ significantly increases in cirrhosis liver parenchyma in comparison with the control group. The results obtained suggest that alpha 1-adrenergic receptors play an important role in cirrhosis formation in children, showing liver parenchyma affection severity and its regenerative properties.


[Fibrinolytic complexes of low-molecular heparin and acetylsalicylic acid]. Complexes of low-molecular heparin with acetylsalicilic acid was formed in vitro when the weight ratio of components was 1:1, 1:5 and 5:1, respectively. All the complexes possessed fibrinolytic and anticoagulating activities. The complex possessed the highest activity when the ratio of heparin to acetylsalicilic acid was 5:1. This complex at a dose 1 mg/200 g of rat weight had the largest thrombolytic effect on the experimental fresh thrombus.


[The age-related characteristics of the hormonal regulation of Na+, K(+)-ATPase activity in the kidney cortex of rats]. The aldosterone binding in isolated distal convoluted and cortical collecting tubules of renal nephrons and the influence of hormonal induction on the Na, K-ATPase activity in membrane fraction of kidney cortex were studied in 10-day- and 2-month-old rats. No reliable difference in aldosterone-specific binding was revealed (0.26 +/- 0.04 and 0.22 +/- 0.03 fmol/mm of tubule length, respectively, at the age of 10 days and 2 months). It was found that Na, K-ATPase activity increased with age from 0.39 +/- 0.06 to 0.72 +/- 0.10 mumol Pi/mg of protein.1 hour.100 microliters. Aldosterone induction caused approximately a 3-fold increase of the enzyme activity in both age groups comparing to the control level. Co-induction of aldosterone and spironolactone resulted in a 50% decrease of Na, K-ATPase activity in adult rats, but did not influence that in young rats. The revealed age-related differences in the mechanism of hormonal Na, K-ATPase regulation are supposed to underlie the absence of physiological reaction of the kidney to aldosterone in early postnatal ontogenesis.


[A pharmacological analysis of the role of GABA- and opioidergic systems in the development of brain edema]. Opioid receptors blocker naloxone potentiated edematous effect of GABA-receptors blockers in experiments on rats. In combination with bicuculline and picrotoxine that agent leads to increased content of water in the brain and decreased brain tissue density. At the same time naloxone prevented GABA-antagonists activation of oxidative processes in brain mitochondria.


[The expression of plasmalemma transcobalamin-II receptors on human blood lymphocytes stimulated by mitogens]. The influence of mitogens on the expression of surface membrane TC-II receptors of human blood lymphocytes and internalization of (TS-II+57Co-CNCbl) complex into cytoplasm were investigated. Mature lymphocytes have a very small number of surface receptors to plasma TC-II but their expression is increased significantly by PHA or Con-A stimulation. CBl transport to cytoplasma is activated in definite sequence by two different mechanisms. Stimulated cells take free CBl without participation of TC-II in early hours of mitogen action (12-42 hrs) before maximal 3H-thymidine incorporation into DNA. On day 3 of cultivation, specific mechanism of CBl transport triggers and the number of lymphoblast receptors is increased manifold. Radioactive CBl enters cytoplasma due to interaction of TC-II-CN [57Co] CBl of the medium with surface membrane receptor of the cells. Thus, the definition of TC-II receptors as an important functional parameter may serve a marker of proliferating cells.


[The ionic regulation of the receptor binding of S35-tert-butylbicyclophosphorothionate by brain membranes in inbred mice with different emotional stress reactions]. Comparative study of effects of ionic concentrations on [35S]-TBPS binding was performed on brain membranes of inbred mice. It was shown that low concentrations of LiCl (less than 100 mM) were more stimulatory effective in Balb/c (C) mice than in C57Bl/6 (B6) mice. On the contrary, stimulatory activity of NaNO (less than 100 mM) was more pronounced in B6 mice. The revealed line of activity of cations (Li+ > K+ > Na+) and anions (Cl- > or = Br- > I-) makes it possible to doubt interpretation of the obtained data only as affinity of the ions to their channels. We suggest the role of ions as modifiers of surface charge of neuronal membrane, that may be the main cause of the revealed interstrain differences.


[The molecular cloning of a genetic region determining the surface exclusion system of the F-like plasmid pAP42]. Molecular cloning of genetic region of F-like plasmid pAP42, coding its surface exclusion system (system Six V) was performed. Restriction and genetic analysis of recombinant plasmids showed that six V locus is situated in Sal I-fragment f5 (4.2 MD) of this plasmid.


Acquisition of interleukin-3 independence in FDC-P2 cells after transfection with the activated c-H-ras gene using a bovine papillomavirus-based plasmid vector. Since the ras family of proto-oncogenes is supposed to be involved in leukemogenesis by point-mutational activation, we studied the effect of the activated ras gene on the growth of a murine interleukin-3 (IL-3)-dependent cell line, FDC-P2. The human activated c-H-ras gene was transfected into FDC-P2 cells by electroporation using a high-level expression vector, BMGhph, which contains a partial DNA sequence from bovine papillomavirus (BPV) and a hygromycin B (hmB)-resistant gene as a selectable marker. The transformed FDC-P2 cells showed a high incidence of IL-3-independent growth and tumorigenicity in nude mice. These clones did not express or secrete IL-3, suggesting the acquisition of IL-3 independence by a nonautocrine mechanism. The high incidence of autonomous growth may be due to the use of the BMG vector, because (1) the activated ras gene in pBR322 vector (pHs-49) was not so efficient in the induction of IL-3 independence, (2) the c-H-ras genome copies per cell increased in number up to about 50 copies by using the BMG vector, and (3) cotransfection with the activated ras gene and the BPV gene in separate plasmids partly enhanced the incidence of autonomous growth without increasing the copy number of the ras gene compared with transfection with the activated ras gene alone. The present study supports the idea that the activation of ras gene is an important step in malignant transformation of hematopoietic cells and suggests that the BPV gene products may cooperate with ras gene activation probably by affecting the cellular genes that may be involved in multistep leukemogenesis. The BMG vector may be useful to test the transforming ability of oncogenes whose oncogenic potential is relatively low.


Influenza A virus binding to human neutrophils and cross-linking requirements for activation. Although neutrophils are not viewed as a principal defense against influenza A virus (IAV) infection, their interactions are both complex and clinically relevant. Activation of the neutrophil is distinctive from that described for chemoattractants. To more fully characterize the pathway by which IAV stimulates the human neutrophil, we have examined its binding characteristics. First, inhibition studies with various sialic acid-containing and sialic-free sugars showed that IAV binds to sialic acid residues and activates receptors distinct from those used by Concanavalin-A (Con-A) and formyl-methionyl-leucyl-phenylalanine (FMLP) and that overlap those bound by wheat germ agglutinin (WGA). That viral hemagglutinin (HA) mediates viral binding and activation was shown by preincubating neutrophils with purified monovalent bromelain-released HA (BHA) and showing that IAV-induced membrane depolarization and hydrogen peroxide (H2O2) production were inhibited approximately 95%. However, binding inhibition required significantly higher concentrations of purified HA, suggesting that binding and cell activation have different interactive requirements. Desialation of the neutrophil surface membrane by neuraminidase treatment resulted in a 90.6% +/- 4.4% and 53.1% +/- 8.7% inhibition of IAV activation of neutrophils and viral binding, respectively. Resialation with ganglioside GT1b totally restored viral binding, but did not reverse the inhibition of activation. Thus, although HA was shown to mediate binding and neutrophil activation, viral binding per se was insufficient to stimulate the cell. Having demonstrated the functional role of HA, we sought to establish the mechanism of stimulation. HA in three different forms (BHA, HA-rosettes, and HA-liposomes) failed to activate the cell, although H2O2 production evoked by IAV stimulation was reduced in competitive inhibition studies with each preparation. Upon cross-linking with a monoclonal antibody to HA, activation comparable to that of intact virus was observed. The requirement for cross-linking of functional receptors, as opposed to activation through the neutrophil Fc receptor, was confirmed in experiments using staphylococcal A protein. These studies have shown the chemical specificity of IAV binding to the human neutrophil, the character of the receptor(s) stimulated to activate the IAV-evoked response, and the activation requirement for cross-linking those receptors responsible for stimulating functional responses.


Interleukin-8 is constitutively and commonly produced by various human carcinoma cell lines. We examined the production of interleukin-8 and interleukin-6 by 30 human carcinoma cell lines. Serum levels of interleukin-8 were measured in 14 patients with hepatocellular carcinoma by enzyme-linked immunosorbent assay and Northern blotting. Furthermore, serum interleukin-8 was also investigated in a nude mouse bearing a tumor of the HuH7 hepatoma cell line producing interleukin-8. Of the 30 cell lines, 29 (96.7%) constitutively produced interleukin-8, and 19 of the 29 (65.5%) were high producers (> 1 ng/ml culture supernatant). Among the high producers, 4 cell lines released both interleukin-8 and interleukin-6. Interleukin-6 was constitutively produced by 17 of the 30 (56.7%) cell lines, 4 of which (23.5%) were high producers (> 1 ng/ml). By Northern blot analysis, mRNAs of interleukin-8 and interleukin-6 were detected in producing cell lines. Of 14 patients with hepatocellular carcinoma 4 (28.5%) showed increased levels of serum interleukin-8. Furthermore, inoculation of the HuH7 hepatoma cell line which produced the highest amount of interleukin-8 into a nude mouse resulted in tumor production accompanied by an elevated level of human interleukin-8 (646 pg/ml) in the peripheral blood. Thus, interleukin-8 is constitutively and commonly produced by various carcinoma cell lines. The production of interleukin-8 by carcinoma cells may be related to the elevation of serum interleukin-8 in patients with hepatocellular carcinoma. Finally, these cell lines may be valuable for studying the relationship between interleukin-8 and cancer.


Adenosine receptors on human leukocytes. IV. Characterization of an A1/Ri receptor. Adenosine (10(-9)-10(-6) mol/l) and R-phenylisopropyladenosine (10(-9)-10(-7) mol/l) partially inhibited the intracellular accumulation of cyclic AMP induced by isoproterenol, prostaglandin E1, histamine and 5'-N-ethylcarboxamidoadenosine in lymphocytes. In contrast, S-phenylisopropyladenosine, which is a poor agonist of the adenosine A1/Ri receptor, had essentially no inhibitory effect. 8-Phenyltheophylline, in low concentrations that do not inhibit cyclic AMP phosphodiesterase, completely blocked the inhibitory effect of R-phenylisopropyladenosine on the increase in cyclic AMP induced by prostaglandin E1. R-Phenylisopropyladenosine (10(-8)-10(-6) mol/l) also inhibited the cyclic AMP accumulation in lymphocytes induced by forskolin (10(-5) mol/l), which activates adenylate cyclase through direct interaction with the enzyme. We also investigated the presence of the adenosine A1/Ri receptor on human polymorphonuclear leukocytes. R-Phenylisopropyladenosine (3 x 10(-9)-10(-7) mol/l) abolished the stimulating effects of prostaglandin and forskolin on cyclic AMP accumulation in polymorphonuclear leukocytes. This effect was blocked by 8-phenyltheophylline and was not observed with the stereoisomer S-phenylisopropyladenosine. The results support the existence of an A1/Ri receptor that regulates cyclic AMP metabolism of human lymphocytes and polymorphonuclear leukocytes.


Optimizing signal and mass resolution for matrix-assisted laser desorption utilizing a linear time-of-flight mass spectrometer. Results are presented for various instrumental configurations employed for matrix-assisted laser desorption mass spectrometry. Mass resolution is determined for a linear time-of-flight mass spectrometer for various lengths of the field-free region. A wire ion guide is utilized and is shown to improve ion transport efficiencies for longer field-free regions. It is also determined experimentally that a modest mass resolution increase is often obtained in configurations employing the wire ion guide when compared to the mass resolution obtained with the same geometry without the wire ion guide. Optimal applied potentials are determined for the wire ion guide. No mass dependence on the optimal applied potential (-100 V) for the wire ion guide is observed for samples of equine myoglobin (MW 16,951.5 Da) and a bacterial protease (MW 27,228.4 Da). The optimal applied voltage was also found to be identical (-100 V) for the singly through quadruply charged molecular ion species of rabbit gamma globulin (MW approximately 150,000 Da). It is shown that a 2 m flight tube with a wire ion guide provides better signal-to-noise mass spectra than a 1 m flight tube without the wire ion guide and can more than double the mass resolution obtainable. Utilization of a 4 m flight tube gives minimal mass resolution enhancement at the expense of signal-to-noise.


Retinoic acid receptor beta 2 mRNA is elevated by retinoic acid in vivo in susceptible regions of mid-gestation mouse embryos. Many of the biological effects of retinoic acid are mediated by its nuclear receptors (RAR-alpha, RAR-beta, and RAR-gamma), and each of these three receptors exist in multiple isoforms. As a first step to identify if any of the receptor isoforms are involved in dysmorphogenesis which is induced in mouse embryos after treatment with retinoic acid (RA), we examined the levels of mRNA of several isoforms of each RAR in the limb buds and other embryonic regions of normal and RA-treated embryos. Within 3 to 6 hr after treatment of mice on day 11 of gestation with RA, RAR-beta 2 mRNA levels in the whole embryo increased 7-fold while both RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated only 2-fold. Since RA treatment of day 11 embryos especially produces limb defects in virtually every embryo, we next examined individual embryonic regions separately. Limb buds showed the highest elevations in RAR-beta 2 mRNA levels (12-fold) compared to a moderate elevation in the head/craniofacial region (8-fold) and a small elevation in the remainder of the body (4-fold). In contrast, RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated in all these tissues to a similar extent, which amounted to only about a 2-fold increase. Retinol, the precursor of RA in the embryo, was also capable of elevating RAR-beta 2 mRNA levels in the limb bud, but the increase was delayed, apparently indicating that metabolic conversion of retinol to RA preceded the effect on mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrastructural study of foetal and adult mouse renal tissue implanted in testis. Kidneys of mice foetuses 15, 17, 19 days old, as well as kidneys of mice 1 and 4 weeks old of the pure C3H/SY species, were implanted into the right testis of 40 adult mice 1.5 to 2 months old, of the same species. The animals were sacrificed after 30 and 60 days and the evolution and development of the renal implants within the testis ware studied. The findings in the light as well as in the electron microscope, showed that the renal implants presented the histologic characters of nephroblastoma, which became clearer the more prolonged the time of the implantation was.


Histological grading of gliomas.

Histological grading of gliomas. Usefulness of grading systems based on non-specific features (e.g. mitosis, necrosis) is unequivocal in only certain types of astrocytomas, and their efficiency remains to be seen in oligodendrogliomas and in mixed oligo-astrocytomas. The possibility of grading ependymomas is still uncertain. Accordingly, only a small proportion of gliomas which occur in children can be reliably graded. Lack of reproducibility is a severe impediment of both current conventional and kinetic methods in the prognosis of gliomas.


[Combination of chemotherapy (vindesine, lomustine, cisplatin, cyclophosphamide) and thoracic radiotherapy in nonresectable non-small cell bronchial carcinoma: final results of a phase II clinical trial]. Seventy-five patients with locally advanced non small cell lung carcinoma were entered in a phase II study combining chemotherapy (vindesine, lomustine, cisplatin and cyclophosphamide) and radical thoracic radiotherapy delivering a total dose of 60-65 Gy. Patients were regularly assessed by radiological and fiberoptic bronchoscopy examinations in order to evaluate local control. An objective response was observed in 22 patients (29%) after initial chemotherapy (2 complete remissions and 20 partial responses). The complete response rate after the combined schedule was 30%. Toxicity of this combination was acceptable. Median survival was 13.5 months. Actuarial risk of developing distant metastases at 3 years was 60%. However, the main cause of failure was local with 80% of uncontrolled or recurrent thoracic tumor in the first 2 years of follow-up. The present study shows that local control remains a major problem in the management of patients with inoperable non metastatic non small cell lung cancer.


[Mechanism of action of retinoids in a new therapeutic approach to acute promyelocytic leukemia]. Vitamin A (retinol) and retinoic acid, its natural derivative, play an important role in the growth, differentiation and development of known normal tissues. Retinoids have recently become of interest to research in areas as diverse as dermatology, embryonal development and cancer research. Retinol is the major retinoid transported in the blood and tissues by its specific carrier retinol binding protein (RBP). The normal level of retinol in plasma is regulated very precisely by retinol homeostasis. RBP-retinol circulation supplies target cells, which then activate retinol into retinoic acid (RA) if they possess the NAD-dependent enzymatic oxidation system. RA, which is one of the most active metabolites of retinol, is also present in low concentration in the blood and the RA rate formation varies from tissues depending on specific need of the cell. The cellular transport and biological activity of retinoids may be mediated by their specific cytoplasmic binding proteins cellular retinol binding protein (CRBP) and the cellular retinoic acid binding protein (CRABP) which may function as shuttles targetting RA to nucleosol fraction and/or as regulator of cellular concentration of RA. The nuclear proteins RARs (retinoic acid receptors), which are members of the nuclear receptor superfamily are likely to be the final transducers of the RA signal at the gene expression. All-trans retinoic acid (ATRA) is able to specifically differentiate the malignant cells from leukemic patients with APL in short-term culture. For this reason, APL patients were successfully treated with ATRA (Chinese and French results). Acute promyelocytic leukemia M3 (French-American-British FAB classification) is a rare disease (10% of AML), characterized by a reciprocal chromosome 15-17 translocation. It has been shown that the chromosome 17 breakpoint of the translocation is localized within the RAR alpha gene. Due to the t(15;17) RAR alpha gene translocated to a gene PML on chromosome 15 resulting in synthesis of PML/RAR alpha fusion messenger RNA. Detection of PML/RAR alpha transcript is now a molecular marker of the disease. The abnormal PML/RAR alpha protein exhibits altered transcription activation properties when compared with RAR alpha. Clinical trials have demonstrated that ATRA is extremely efficient in inducing complete remission in APL patients. The morphologic finding of maturing elements in the bone marrow and peripheral blood during retinoic acid treatment indicates that the remission is obtained without hypoplasia and suggests that a differentiating mechanism is involved.(ABSTRACT TRUNCATED AT 400 WORDS)


Transgenic maize plants by tissue electroporation. In this paper, we describe the transformation of regenerable maize tissues by electroporation. In many maize lines, immature zygotic embryos can give rise to embryogenic callus cultures from which plants can be regenerated. Immature zygotic embryos or embryogenic type I calli were wounded either enzymatically or mechanically and subsequently electroporated with a chimeric gene encoding neomycin phosphotransferase (neo). Transformed embryogenic calli were selected from electroporated tissues on kanamycin-containing media and fertile transgenic maize plants were regenerated. The neo gene was transmitted to the progeny of kanamycin-resistant transformants in a Mendelian fashion. This showed that all transformants were nonchimeric, suggesting that transformation and regeneration are a single-cell event. The maize transformation procedure presented here does not require the establishment of genotype-dependent embryogenic type II callus or cell suspension cultures and facilitates the engineering of new traits into agronomically relevant maize inbred lines.


Guinea-pig treatment with pertussis toxin suppresses macrophage-dependent bronchoconstriction by fMLP and fails to inhibit the effects of PAF. 1. Bronchoconstriction and thromboxane B2 (TxB2) release following the intra-tracheal administration of the secretagogue N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) to lungs from pertussis toxin-treated guinea-pigs in vivo and in vitro were inhibited as compared to saline-treated animals, under conditions where the responses to PAF were modified less effectively. 2. The cell target accounting for bronchoconstriction by fMLP and for inhibition by pertussis toxin is located in the airways and is probably the alveolar macrophage. Indeed (a) fMLP-induced superoxide anions and TxB2 formation by alveolar macrophages were inhibited by pertussis toxin given in vivo; (b) Gi proteins of membranes from alveolar macrophages were ADP-ribosylated in vivo by pertussis toxin and (c) bronchoconstriction and TxB2 release in response to the intra-tracheal administration of fMLP to lungs from pertussis toxin-treated animals were restored when alveolar macrophages from control guinea-pigs were transferred into the airways of pertussis toxin-treated animals before lung isolation. 3. Pertussis toxin administered to guinea-pigs in vivo, reduced the subsequent TxB2 formation and superoxide anion release by alveolar macrophages stimulated with PAF, but failed to inhibit PAF-induced bronchoconstriction. 4. Formation of TxB2 by alveolar macrophages following the intra-tracheal administration of fMLP accounts for bronchoconstriction and requires pertussis toxin-sensitive Gi proteins. PAF operates via a different mechanism, which is independent of Gi-like protein and involves mediators other than TxB2 and superoxide anions.


Pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase. 1. This paper describes the pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase which is undergoing clinical evaluation. 2. ICI D2138 potently inhibited leukotriene synthesis in murine peritoneal macrophages (IC50 = 3 nM) and human blood (IC50 = 20 nM). In human and dog blood, ICI D2138 did not inhibit thromboxane B2 synthesis at a concentration of 500 microM, thus the selectivity ratio (cyclo-oxygenase: 5-lipoxygenase) was greater than 20,000. In contrast, zileuton (a 5-lipoxygenase inhibitor also undergoing clinical evaluation) exhibited a selectivity ratio of 15-100. 3. ICI D2138 potently and dose-dependently inhibited ex vivo leukotriene B4 (LTB4) synthesis by rat blood with ED50 values of 0.9, 4.0 and 80.0 mg kg-1 p.o. at 3, 10 and 20 h respectively after dosing. Similar activity was observed for inhibition of LTB4 production in a zymosan-inflamed rat air pouch model. Zileuton produced ED50 values of 5 and 20 mg kg-1 at 3 and 10 h respectively. 4. Oral administration of 1, 3 or 10 mg kg-1 ICI D2138 to dogs produced maximal inhibition of ex vivo LTB4 synthesis by blood for 5, 9 and 31 h respectively. A dose of 5 mg kg-1 p.o. of zileuton caused maximal inhibition of LTB4 for 24 h. 5. Oral administration of 10 mg kg-1 ICI D2138 caused total inhibition of LTB4 production in zymosan-inflamed rabbit knee joint. 6. Topical administration of ICI D2138 to rabbit skin caused a dose-related inhibition of arachidonic acid-induced plasma extravasation with an ID30 of 1.08 nmol per site. Zileuton was approximately 40 times less potent.7. Oral anti-inflammatory activity was assessed in an arachidonic acid-induced mouse ear oedema model in animals treated with indomethacin to block pro-inflammatory prostanoids. ICI D2138, given orally, caused dose-dependent inhibition of oedema with an approximate ID50 of 1.8 mg kg'. Zileuton was approximately 10 times less potent.8. ICI D2138 caused a dose-dependent inhibition of antigen-induced broncho-constriction in guineapigs with an approximate ID50 of 0.1 mg kg-', i.v. Zileuton was approximately 10 times less potent.9. In view of the pharmacological profile described here, ICI D2138 has the potential to provide improved clinical efficacy compared to existing lipoxygenase inhibitors such as zileuton.


Involvement of cyclic GMP in non-adrenergic, non-cholinergic inhibitory neurotransmission in dog proximal colon. 1. Nitric oxide (NO) may serve as a non-adrenergic, non-cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3':5'-cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon. 2. The membrane permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8-Br-cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8-Br-cGMP also inhibited spontaneous phasic contractions. 3. The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity. 4. Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions. 5. Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels. 6. Treatment of muscles with L-NG-nitroarginine methyl ester (L-NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with L-NAME. 7. These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.


Potassium channel openers, NIP-121 and cromakalim, enhance the relaxation induced by sodium nitroprusside in the guinea-pig isolated trachea. 1. The effect of the potassium channel openers, NIP-121 and cromakalim, on agonist-induced relaxation of the guinea-pig isolated trachea was investigated and the results were compared with those in the epithelium-denuded trachea. 2. Tracheal strips were incubated with a potassium channel opener or vehicle for 30 min in the presence of 5 microM indomethacin and then contracted with 30 nM leukotriene D4 (LTD4). Relaxant agents were added to the organ bath after the LTD4-elicited contraction had reached a plateau. 3. In epithelium-intact trachea, NIP-121 0.1 microM and cromakalim 1 microM, which did not modify the LTD4 (30 nM)-induced contraction, significantly enhanced the sodium nitroprusside (SNP)-induced relaxation. This enhancement of relaxation was not seen in the case of relaxation induced by the cyclic AMP-dependent bronchodilators isoprenaline, vasoactive intestinal peptide or prostaglandin E2. The enhancement of SNP-induced relaxation by NIP-121 and cromakalim was abolished in the presence of the ATP-sensitive potassium channel blocker, glibenclamide (1 microM). NIP-121 and cromakalim did not produce any significant changes in the relaxation induced by 8-bromoguanosine-cyclic monophosphate (8-Br-cyclic GMP), a cyclic GMP analogue. 4. In epithelium-denuded trachea, SNP-induced relaxation alone was significantly enhanced but that induced by 8-Br-cyclic GMP was not changed. Neither NIP-121 nor cromakalim enhanced SNP-induced relaxation in denuded trachea.5. These results suggest that in the presence of an intact epithelium the enhancement by NIP-121 and cromakalim of the relaxation of guinea-pig tracheal smooth muscle induced by SNP may be associated with the opening of glibenclamide-sensitive potassium channels.


Bradykinin-induced activation of nociceptors: receptor and mechanistic studies on the neonatal rat spinal cord-tail preparation in vitro. 1. The effects of bradykinin on nociceptors have been characterized on a preparation of the neonatal rat spinal cord with functionally connected tail maintained in vitro. Administration of bradykinin to the tail activated capsaicin-sensitive peripheral fibres and evoked a concentration-dependent (EC50 = 130 nM) depolarization recorded from a spinal ventral root (L3-L5). 2. The response to bradykinin was unaffected by the peptidase inhibitors, bestatin (0.4 mM), thiorphan (1 microM), phosphoramidon (1 microM) and MERGETPA (10 microM) or by the presence of calcium blocking agents, cadmium (200 microM) and nifedipine (10 microM). 3. Inhibition of cyclo-oxygenase with indomethacin (1-5 microM), aspirin (1-10 microM) and paracetamol (10-50 microM) consistently attenuated responses to bradykinin. 4. The effect of bradykinin was mimicked by the phorbol ester PDBu, an activator of protein kinase C. The response to bradykinin was attenuated following desensitization to PDBu but desensitization to bradykinin did not induce a cross-desensitization to PDBu. The protein kinase C inhibitor staurosporine (10-500 nM) consistently attenuated the effects of PDBu and bradykinin. 5. Bradykinin responses were reversibly enhanced by dibutyryl cyclic AMP (100 microM). However dibutyryl cyclic GMP (0.5 mM) and nitroprusside (10 microM) produced prolonged block of responsiveness to bradykinin. Prolonged superfusion with pertussis toxin did not affect responses to bradykinin. 6. The B1-receptor agonist des Arg9-bradykinin (10-100 microM) was ineffective alone or after prolonged exposure of the tail to lipopolysaccharide (100 ng ml-1) or epidermal growth factor (100 ng ml-1) to induce B1 receptors. The BI-receptor antagonist, des Arg9 Leu8-bradykinin (10 JM) did not attenuate the response to bradykinin. A number of bradykinin B2 antagonists selectively and reversibly attenuated the response to bradykinin. The rank order potency was Hoe 140> LysLys [Hyp3,Thi5 8,D-Phe7]-bradykinin> D-Arg[Hyp3, Thi5'8, D-Phe7]-bradykinin = D-Arg[Hyp2,Thi5'8, D-Phe7]-bradykinin.7. These data show that bradykinin produces concentration-dependent activation of peripheral nociceptors in the neonatal rat tail. The responses were unaffected by calcium channel block and were partially dependent on the production of prostanoids. Bradykinin-evoked responses were consistent with the activation of protein kinase C-dependent mechanisms. Cyclic GMP-dependent mechanisms may be involved in bradykinin-receptor desensitization whereas cyclic-AMP dependent mechanisms increase fibre excitability and facilitate bradykinin-induced responses. The effects of bradykinin were mediated by a B2 receptor.


Palmitoyl-DL-carnitine has calcium-dependent effects on cultured neurones from rat dorsal root ganglia. 1. The effects of palmitoyl-DL-carnitine (0.01 to 1 mM) on whole cell voltage-activated calcium channel currents carried by calcium or barium and Ca(2+)-activated chloride currents were studied in cultured neurones from rat dorsal root ganglia. 2. Palmitoyl-DL-carnitine applied to the extracellular environment or intracellularly via the patch solution reduced Ca2+ currents activated over a wide voltage range from a holding potential of -90 mV. Inhibition of high voltage activated Ca2+ channel currents was dependent on intracellular Ca2+ buffering and was reduced by increasing the EGTA concentration from 2 to 10 mM in the patch solution. Barium currents were significantly less sensitive to palmitoyl-DL-carnitine than Ca2+ currents. 3. The amplitude of Ca(2+)-activated Cl- tail currents was reduced by palmitoyl-DL-carnitine. However, the duration of these Cl- currents was greatly prolonged by palmitoyl-DL-carnitine, suggesting slower removal of free Ca2+ from the cytoplasm following Ca2+ entry through voltage-activated channels. 4. Palmitoyl-DL-carnitine evoked Ca(2+)-dependent inward currents which could be promoted by activation of the residual voltage-activated Ca2+ currents and attenuated by intracellular application of EGTA. 5. We conclude that palmitoyl-DL-carnitine reduced the efficiency of intracellular Ca2+ handling in cultured dorsal root ganglion neurones and resulted in enhancement of Ca(2+)-dependent events including inactivation of voltage-activated Ca2+ currents. The activation of inward currents by palmitolyl-DL-carnitine may involve Ca(2+)-induced Ca2+ release from intracellular stores, or direct interaction of palmitoyl-DL-carnitine with Ca2+ stores.


Histamine H3 receptors modulate the release of [3H]-acetylcholine from slices of rat entorhinal cortex: evidence for the possible existence of H3 receptor subtypes. 1. The effect of agents which interact with the histamine H3 receptor on potassium-stimulated tritium release from slices of rat entorhinal cortex preloaded with [3H]-choline is described. We have examined the effects of the selective H3 receptor agonist, (R)-alpha-methylhistamine (RAMH), and a number of H3 receptor antagonists, including the selective compound thioperamide, on the potassium-stimulated release of tritium. 2. In the presence of mepyramine and ranitidine, RAMH (0.01-10 microM) inhibited potassium-stimulated tritium release in a concentration-dependent manner, EC50 = 0.11 microM. The maximum inhibition was approximately 50%. 3. Thioperamide displaced the RAMH concentration-response curve to the right yielding a pKB value of 8.4. There was no change in the maximum response to RAMH. 4. Other H3 receptor antagonists, including impromidine and burimamide, also caused rightwards displacement of the linear portion of the RAMH concentration-response curve. However, phenylbutanoylhistamine and betahistine, which are reported to be relatively potent H3 receptor antagonists, showed very low affinity. 5. Thioperamide (0.001-1 microM) alone enhanced the potassium-stimulated release of tritium in a concentration-dependent manner. Maximum effects were observed at 0.1-1 microM thioperamide, enhancing release by approximately 20%. 6. Results are discussed in terms of the regulatory role of H3 receptors on acetylcholine release and the possible existence of H3 receptor subtypes.


Modulation of barrier function of bovine aortic and pulmonary artery endothelial cells: dissociation from cytosolic calcium content. 1. Barrier function and cytosolic free calcium content [Ca2+]i was measured in monolayers of bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC). 2. Thrombin (1 u ml-1) increased albumin transfer across monolayers of BPAEC but not BAEC, yet induced biphasic increases in [Ca2+]i in both endothelial cell types, consisting of a rapid, initial phasic component which decayed to a lower, more sustained plateau phase. 3. 4 beta-Phorbol 12-myristate 13-acetate (PMA; 0.3-3000 nM) increased albumin transfer across monolayers of BPAEC and BAEC, but had no effect on basal levels of [Ca2+]i in either endothelial cell type. 4. Treatment of BPAEC and BAEC with forskolin (30 microM), an activator of adenylate cyclase, had no effect on resting transfer of albumin, but inhibited that stimulated by PMA (600 nM). It also inhibited the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but enhanced the plateau phase of the associated increase in [Ca2+]i. 5. Treatment of BPAEC and BAEC with either atriopeptin II (100 nM), an activator of particulate guanylate cyclase, or 8 bromo cyclic GMP (30 microM) had no effect on resting or PMA (600 nM)-stimulated transfer of albumin. Both agents did, however, inhibit the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but had no effect on the associated increase in [Ca2+]i. 6. These data suggest a dissociation between the ability of agents that increase or decrease albumin transfer and their effects on [Ca2+]i. Consequently, activation of protein kinase C may be the major stimulus for trans-endothelial transfer of macromolecular solutes. Endothelial barrier function is enhanced by elevation of either cyclic AMP or cyclic GMP content. Cyclic AMP appears to act by inhibiting the actions of protein kinase C, while cyclic GMP may act to inhibit a key step proximal to activation of this enzyme.


Effects of PAF on excitatory neuro-effector transmission in dog airways. 1. Effects of PAF on excitatory neuro-effector transmission in smooth muscle cells of mucosa-free trachea and epithelium-intact bronchiole of the dog were investigated, by isometric tension recording, microelectrode and double sucrose-gap methods. 2. PAF (10(-11)-10(-7) M) dose-dependently enhanced the amplitude of contraction evoked by repetitive field stimulations (10 stimuli at 20 Hz) in both tracheal and bronchiolar tissues. At higher concentrations PAF (> 10(-8) M) increased the amplitude of contraction to a greater extent in the bronchiole than in the trachea. 3. In both muscle tissues, in parallel to the amplitude of contraction, PAF markedly enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by a single field stimulation in a dose-dependent manner, with no change in the resting membrane potential or input membrane resistance of the smooth muscle cells. PAF (5 x 10(-7) M) enhanced the amplitude of e.j.p. to a greater extent in the bronchiole than in the trachealis. In contrast, lyso-PAF (10(-10)-10(-7) M) showed no effect on e.j.p. amplitude in bronchiolar tissues. At a high concentration (10(-7) M) lyso-PAF slightly enhanced the e.j.p. amplitude in tracheal tissue, however the lyso-PAF induced stimulation of e.j.p. amplitude in the trachea was small compared to that of PAF. 4. PAF (10(-7) M) had no effect on the membrane depolarization induced by acetylcholine (ACh, 10(-9)-10(-5) M) and carbachol (10(-9)-10(-5) M) in tracheal smooth muscle cells. 5. The PAF-antagonists CV3988 (5 x i0-7 M) or WEB2086 (5 x 10-7 M) significantly enhanced the e.j.p. amplitude themselves, PAF (5 x 10-8 M) further enhanced the ej.p. amplitude in the presence of WEB2086 (5 x l0-7 M) but not CV3988 (5 x 10-7 M). In contrast, the new PAF-antagonist, E 6123(5 x l0-8 M), did not affect the ej.p. amplitude itself, and completely inhibited the increase in ej.p. amplitude caused by 5 x 10-8 M PAF. On the other hand, in the presence of the Hi-antagonist,mepyramine, PAF (5 X 10-8 M) further enhanced the ej.p. amplitude.6. The leukotriene synthesis inhibitor AA-861 (10-6 M) or leukotriene antagonist ONO1078 (10-7 M)inhibited the increase in ej.p. amplitude caused by 5 X 10-8 M PAF, respectively.7. In the presence of AA-861 (10-6 M), leukotriene B4 (LTB4, 10-' M) or LTD4 (10-8 M) slightly, and LTC4 (10- M) markedly enhanced the ej.p. amplitude. In contrast, LTE4 (10-8 M) significantly suppressed the e.j.p. amplitude.8. PAF (5 x 10-8 M) attenuated the depression phenomena of ej.ps observed during double stimulus experiments at different time intervals (5-10 s), but had no effect on the summation of ej.ps during repetitive field stimulation at a high frequency (20 Hz) in the trachealis.9. These results indicate that PAF potentiates excitatory neuro-effector transmission mainly through stimulating the release of lipoxygenase products, mainly LTC4 in the dog airway smooth muscle tissues.


Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. 1. Angiotensin converting enzyme (ACE)-inhibitors have been demonstrated to be effective in the treatment of cardiac hypertrophy when used in antihypertensive doses. The aim of our one year study with an ACE-inhibitor in rats was to separate local cardiac effects produced by a non-antihypertensive dose from those on systemic blood pressure when an antihypertensive dose was used. 2. Rats made hypertensive by aortic banding were subjected to chronic oral treatment for one year with an antihypertensive dose of the ACE inhibitor, ramipril 1 mg kg-1 daily, (RA 1 mg) or received a low dose of 10 micrograms kg-1 daily (RA 10 micrograms) which did not affect high blood pressure. 3. Chronic treatment with the ACE-inhibitor prevented left ventricular hypertrophy in the antihypertensive rats as did the low dose which had no effects on blood pressure. Similar effects were observed on myocardial fibrosis. Plasma ACE activity was inhibited in the RA 1 mg but not in the RA 10 micrograms group although conversion of angiotensin (Ang) I to Ang II in isolated aortic strips was suppressed in both treated groups. Plasma catecholamines were increased in the untreated control group, but treatment with either dose of ramipril normalized the values. The myocardial phosphocreatine to ATP ratio (an indicator of the energy state in the heart) was reduced in the vehicle control group whereas the hearts from treated animals showed a normal ratio comparable to hearts from sham-operated animals. 4. After one year, five animals were separated from each group, treatment withdrawn, and housed for additional six months. In the RA 1 mg group, blood pressure did not reach the value of the control vehicle group and surprisingly, left ventricular hypertrophy and myocardial fibrosis did not recur in animals during withdrawal of treatment.5. These data show that long term ACE inhibitor treatment with ramipril in antihypertensive and non-antihypertensive doses prevented cardiac hypertrophy and myocardial fibrosis. This protective effect was still present after 6 months treatment withdrawal.


Role of the L-arginine-NO pathway and of cyclic GMP in electrical field-induced noradrenaline release and vasoconstriction in the rat tail artery. 1. The possible roles of the L-arginine-NO pathway and of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in regulating the prejunctional release of noradrenaline and neurogenic vasoconstriction were investigated in the perfused rat tail artery. 2. In the presence of N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of NO formation, the vasoconstrictor responses to perivascular nerve stimulation (24 pulses at 0.4 Hz, 0.3 ms, 200 mA) and to exogenous noradrenaline (1 microM) were significantly enhanced, whereas the stimulation-evoked tritium overflow from [3H]-noradrenaline preloaded arteries was not modified. The vasoconstriction enhancing effect of L-NAME was prevented by L-arginine (1 mM) but not D-arginine (1 mM) and was abolished by removal of the endothelium. 3. The NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1; 0.1-30 microM), and the cyclic GMP phosphodiesterase inhibitor, zaprinast (0.1-30 microM) both induced a concentration-dependent inhibition of the electrical field stimulation-induced vasoconstriction, while atrial natriuretic peptide (ANP; 100 nM) produced only a slight decrease of the vasoconstrictor response. Methylene blue (3 microM), a known inhibitor of soluble guanylate cyclase increased the electrical field stimulation-induced vasoconstriction. SIN-1 and methylene blue when administered simultaneously, antagonized each others effect. None of the compounds tested (SIN-1, zaprinast, ANP or methylene blue) had any significant effect on the stimulation-evoked [3H]-noradrenaline overflow. 4. 8-Bromo-cyclic GMP, a potent activator of cyclic GMP-dependent protein kinase, markedly and concentration-dependently (3-300 microM) increased [3H]-noradrenaline overflow but decreased field stimulation-induced vasoconstriction. Dibutyryl-cyclic GMP (100 JM), a weak activator of cyclic GMP-dependent protein kinase, affected neither the pre- nor the postjunctional response to electrical field stimulation.5. These data show that an NO-like substance of endothelial origin, derived from L-arginine, attenuates vasoconstriction in the rat tail artery, whether neurally-induced or evoked by exogenous noradrenaline.Since noradrenaline release was unaltered by compounds modifying NO production, this NO-like compound acted through a postjunctional mechanism. The lack of prejunctional effects of both soluble and membrane-associated guanylate cyclase activators, despite a large effect of 8-bromo-cyclic GMP,suggests that endogenous cyclic GMP production, if present in sympathetic nerves, may not be involved in the regulation of noradrenaline release in the rat tail artery.


BK1 and BK2 bradykinin receptors in the rat duodenum smooth muscle. 1. The dual action of bradykinin (relaxation and contraction) on the rat duodenum was investigated by studying its effect on adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultured duodenal smooth muscle cells, and the effects of apamin on the isolated muscle responses to agonists and antagonists of BK1 and BK2 receptors. 2. No change was observed in the cyclic AMP content of cultured cells incubated with up to 100 nM bradykinin. 3. Apamin (100-500 nM) inhibited the relaxant component and enhanced the contractile component of the responses to bradykinin and to the BK2-specific analogue [Thi5,8,D-Phe7]-bradykinin. 4. Apamin (100-500 nM) did not affect the contractile response of stretched duodenum preparation to the BK1-specific agonist des-Arg9-bradykinin. 5. The BK2 antagonist, [D-Arg0Hyp3Thi5,8,D-Phe7]-bradykinin, at a concentration which completely inhibited the relaxant response to bradykinin and to [Thi5,8,D-Phe7]-bradykinin, also prevented the contraction in response to either agonist in the presence of apamin. 6. Our results demonstrate two populations of bradykinin receptors in rat duodenum: a BK2 subtype responsible for the biphasic response of the non-stretched duodenum, and a BK1 subtype responsible for the contractile effect on the stretched tissue.


Rectal cancer risk in older patients with familial adenomatous polyposis and an ileorectal anastomosis: a cause for concern. The risk of rectal cancer in 224 patients with familial adenomatous polyposis with an ileorectal anastomosis has been estimated by life-table analysis. Until the age of 50 years the cumulative risk is reasonably low at 10 per cent (95 per cent confidence interval 4.5-16 per cent), increasing sharply to 29 per cent (95 per cent confidence interval 18-40 per cent) by the age of 60 years. This means that surveillance of the retained rectum in older patients must either be improved or they should undergo restorative proctocolectomy in earlier middle age.


Cl(-)-mediated interaction between GABA and glycine currents in cultured rat hippocampal neurons. A cross-interaction between GABA- and glycine-evoked currents was found when the two transmitters were applied in sequence to cultured embryonic rat hippocampal neurons. Whole-cell GABA-current was inhibited by a previous glycine-current flowing in the same direction (inward or outward), and potentiated by a current with opposite polarity. The same effect was caused by GABA on glycine-current. Repeated applications of GABA (glycine) elicited currents of decreasing or increasing amplitude, according to a similar pattern. Transmitter interaction was independent of external Ca2+ and of all the metabolic pathways tested, but it was blocked by specific receptor antagonists, bicuculline and strychnine. The extent of both inhibition and potentiation correlated with the amount of charge flowing through the membrane during the conditioning transmitter application, indicating that cross-modulation depends on shifts of Cl- reversal potential. This finding has both functional and methodological implications, as it suggests a new mechanism of transmitter interaction in the brain, and also that patch-clamp pipettes cannot adequately perfuse cell interior.


Effects of N-methyl-D-aspartate on quisqualate-stimulated phosphoinositide hydrolysis in slices of kitten striate cortex. Stimulation of phosphoinositide (PI) hydrolysis by excitatory amino acids (EAAs) was studied in coronal slices of kitten visual cortex. Coincubation with N-methyl-D-aspartate (NMDA) markedly reduced the stimulation by quisqualate, however, this inhibition developed with a latency of > 10 min and occurred even when the NMDA exposure preceded, but did not overlap with, incubation in quisqualate. This time-course of NMDA inhibition of EAA-stimulated PI turnover places new constraints on its possible mechanism of inhibition.


Localization of striatal excitatory amino acid binding site subtypes to striatonigral projection neurons. Quantitative autoradiography was used to examine the cellular localization of excitatory amino acid binding sites in the striatum following selective lesion of striatonigral projection neurons. Degeneration of striatonigral neurons was induced unilaterally by injection of the suicide transport toxin, volkensin, into the left substantia nigra. Twelve days following nigral volkensin injection there was a reduction of all excitatory amino acid binding site subtypes in the striatum ipsilateral to the injected nigra. The reduction in N-methyl-D-aspartate (NMDA) binding sites was significantly greater than the loss of D,L-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA), kainate and metabotropic binding. These results indicate that there are NMDA, AMPA, metabotropic and kainate binding sites on striatonigral projection neurons and suggest that the NMDA subtype may be selectively enriched on striatonigral neurons.


The stimulation of central kappa opioid receptors decreases male sexual behavior and locomotor activity. Systemic injections of the kappa (kappa) opioid receptor agonist U-50,488H decreased male sexual behavior, locomotor activity, body temperature and bodily grooming, and induced body flattening. The U-50,488H-induced inhibitions of male sexual behavior were prevented by systemic injections of naloxone and by intra-cranial injections of the kappa opioid antagonist nor-binaltorphimine (NBNI). Injections of NBNI to either the ventral tegmental area (VTA) or the nucleus accumbens septi (NAS) increased female-directed behavior, and prevented the U-50,488H-induced decreases in female-directed behavior. Intra-VTA NBNI prevented U-50,488H-induced decreases in the mean number of ejaculations, intra-NAS NBNI prevented U-50,488H-induced increases in copulation latencies. Intra-medial preoptic area (mPOA) injections of NBNI increased female-directed behavior, and attenuated U-50,488H-induced decreases in female-directed behavior as well as U-50,488H-induced increases in both copulation and ejaculation latencies. Injections of NBNI dorsal to the mPOA were ineffective. Two of 26 days following the central injection of NBNI, systemic injections of U-50,488H remained behaviorally ineffective, leaving both sexual behavior and locomotor activity undiminished. These results suggest that the stimulation of central kappa opioid receptors inhibits sexual behavior in the male rat; perhaps endogenous kappa opioid agonists induce sexual refractory periods.


Parallel activation of multiple spinal opiate systems appears to mediate 'non-opiate' stress-induced analgesias. Pain is powerfully modulated by circuitries within the CNS. Two major types of pain inhibitory systems are commonly believed to exist: opiate (those that are blocked by systemic opiate antagonists and by systemic morphine tolerance) and non-opiate (those that are not). We used intrathecal delivery of mu, delta, and kappa opiate receptor antagonists to examine 3 well-accepted non-opiate stress-induced analgesias. Combined blockade of all 3 classes of opiate receptors antagonized all of the 'non-opiate' analgesias. Further experiments demonstrated that blocking mu and delta or mu and kappa was sufficient to abolish 'non-opiate' analgesias. Combined blockade of kappa and delta receptors was without effect. The clear conclusion is that all endogenous analgesia systems may in fact be opiate at the level of the spinal cord. Phenomena previously thought to be non-opiate appear to involve parallel activation of multiple spinal opiate processes. These findings suggest the need for a fundamental shift in conceptualizations regarding the organization and function of pain modulatory systems in particular, and opiate systems in general.


Opioid mu-deficient CXBK mouse and the role of mu 1-receptors in electrically induced convulsions. Studies were made on the role of mu 1-receptors on electrically induced convulsions in mice. A relatively selective mu 1-agonist, DAGO ([D-Ala2-N-methyl-Phe4-Gly-ol]-enkephalin) decreased the durations of tonic and clonic convulsions in C57BL/6 mice, but not in opioid mu-deficient CXBK mice, indicating that the activation of mu 1-receptors had an anticonvulsive effect on these convulsions in mice. The selective mu 1-antagonists naloxonazine and naltrexonazine promoted the clonic phase of electrically induced convulsion in C57BL/6 mice. Moreover, the duration of the clonic phase was longer in mu-deficient CXBK mice than in C57BL/6 mice. These data for CXBK mice confirmed, at least some proconvulsant effects of mu 1-antagonists in C57BL/6 mice. Thus, blockade of mu 1-receptors has a proconvulsant effect, and mu 1-receptors have at least some protective role against electrically induced convulsions.