Granulocyte-colony stimulating factor stimulates immunoreactive endothelin-1 release from cultured bovine endothelial cells. We examined the effect of human recombinant granulocyte-colony stimulating factor (G-CSF) on the release of immunoreactive endothelin-1 (ET-1) from cultured bovine vascular endothelial cells. G-CSF dose dependently (10(-8)-10(-6) M) increased the release of immunoreactive ET-1 as a function of time under a serum-free condition. Coaddition of G-CSF and thrombin induced an additive effect on immunoreactive ET-1 release. Neither Ca2+ channel antagonist nor cyclooxygenase inhibitor affected immunoreactive ET-1 release stimulated by G-CSF. These results suggest that G-CSF, in addition to its effect on granulocyte progenitors, has a direct effect on vascular endothelium to induce the release of immunoreactive ET-1.


Delta-opioid-receptor activation by [D-Pen2,D-Pen5]enkephalin and morphine inhibits substance P release from trigeminal nucleus slices. The release of substance P (SP) from spinal dorsal horn slices is partially inhibited by micromolar concentrations of selective delta-opioid receptor agonists. In the present study, we have examined the effect of nanomolar concentrations of [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-opioid receptor agonist) and low micromolar of concentrations morphine on K(+)-evoked SP release from rat trigeminal nucleus caudalis (TNC) slices. DPDPE and morphine inhibited SP release with an apparent maximal effect at 3 nM and at 3 microM, respectively. DPDPE and morphine produced U-shaped concentration-response curves that were completely autoinhibited at 100 nM DPDPE and 1 microM morphine. The inhibition of SP release produced by 3 nM DPDPE and 3 microM morphine was blocked by the opioid receptor antagonists naloxone (30 nM; non-selective) and ICI 174,864 (0.3 microM; delta-selective) but not by nor-binaltorphimine (3 nM n-BNI; kappa-selective), naloxonazine (1 nM; micro 1-selective) or beta-funaltrexamine (20 nM beta-FNA; mu-selective). These findings indicate that delta-opioid receptor-mediated inhibition of SP release from TNC can be achieved by nanomolar concentrations of selective delta-opioid receptor agonists. Activation of delta-opioid receptors by morphine might be involved in the residual analgesia observed after mu 1-opioid receptor blockade and in the analgesia produced by high doses of morphine.


Fluvoxamine preferentially increases extracellular 5-hydroxytryptamine in the raphe nuclei: an in vivo microdialysis study. The effects of systemic administration of fluvoxamine on extracellular serotonin (5-hydroxytryptamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the frontal cortex and raphe nuclei of freely moving rats were examined. Fluvoxamine significantly increased extracellular 5-HT concentrations in both regions at the two doses used (1 and 10 mg/kg i.p.). However, the increase in the raphe nuclei was several-fold that in the frontal cortex. Dialysate 5-HIAA concentrations decreased after treatment with fluvoxamine. These results confirm that 5-HT uptake inhibitors preferentially increase extracellular concentrations of 5-HT in the vicinity of cell bodies and dendrites of serotonergic neurones.


CR 2039, a new bis-(1H-tetrazol-5-yl)phenylbenzamide derivative with potential for the topical treatment of asthma. The pharmacological activity of CR 2039 (4-(1H-tetrazol-5-yl)-N-(4-[1H-tetrazol-5-yl]phenylbenzam ide)) a newly discovered antiallergic compound is described. CR 2039 administered i.m. or i.v. inhibited rat passive cutaneous anaphylaxis (PCA) with an ED50 of 0.1 mg/kg and a potency about 15 times higher than that of disodium cromoglycate (DSCG). CR 2039 i.m., by aerosol or as dry powder insufflation, gave dose-related significant protection against IgE-dependent bronchial anaphylaxis induced by aerosolized antigen in anesthetized guinea-pigs. In conscious guinea-pigs CR 2039 given i.m. delayed dose dependently (ED50, 17 mg/kg) the onset of bronchoconstriction induced by aerosolized antigen, while DSCG was ineffective up to 100 mg/kg. The protection was accompanied by significant inhibition of the vascular permeability provoked by antigen challenge in all airway segments except trachea. CR 2039 (10-100 mg/kg i.v.) inhibited the microvascular permeability changes in a model of allergic conjunctivitis in sensitized guinea-pigs. CR 2039 inhibited dose dependently guinea-pig lung cAMP-phosphodiesterase with an IC50 of 50 microM.


Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843. The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).


Cyclosporine is angiostatic.

Cyclosporine is angiostatic. The systemic effect of the immunosuppressive drug cyclosporine (CS) on formation of new blood vessels was studied quantitatively in rats using the mesenteric-window assay. Angiogenesis was induced by i.p. injection of saline. CS at a s.c. dose of 4 mg/kg/day, which is in the range used clinically, suppressed angiogenesis (inhibiting branching or tortuosity more than spatial expansion), and appeared to be non-toxic. This is the first report on an apparently selective angiostatic effect of CS. The finding is likely to have implications for the clinical use of CS, not only in certain types of organ transplantation but possibly also in psoriasis and other angiogenesis-dependent diseases.


Differential effects of granulocyte- and granulocyte-macrophage colony-stimulating factors (G- and GM-CSF) on neutrophil adhesion in vitro and in vivo. A direct comparison of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) effects on neutrophil adhesiveness has been carried out. In vitro, GM-CSF and G-CSF upregulate neutrophil CD11b to a similar degree (to 227 +/- 69%, and 232 +/- 70% of control cells, respectively, p < 0.0005), but GM-CSF is more effective in downregulating neutrophil leucocyte adhesion molecule-1 (LAM-1), reducing levels to 33 +/- 4% (p < 0.0005), while G-CSF causes a fall to only 65 +/- 17% (p < 0.005) of control. The concentration of GM-CSF needed to achieve maximal activity is at least one log less than that of G-CSF. In vivo, both GM-CSF and G-CSF upregulate neutrophil CD11b (to 296 +/- 45% and 370 +/- 150%, respectively of baseline), but surface levels of LAM-1 on circulating cells are unchanged. GM-CSF increased neutrophil adhesion to cultured human endothelium in vitro (from 9.3 +/- 0.7% to 15.4 +/- 1.3%, p < 0.0005, n = 10), while G-CSF was without effect. In vivo, both GM-CSF and G-CSF produce a transient leucopenia, but recovery of peripheral counts occurs much earlier (by 60 minutes) with G-CSF, than with GM-CSF (only 50% of cells have demarginated at 120 min). GM-CSF appears to be greater proadhesive agonist for neutrophils than G-CSF.


Interaction of trans-acting factors with the proximal promoter of the mouse alpha-fetoprotein gene. 1. The alpha-Fetoprotein (AFP) gene is expressed during fetal life, but not in adult cells. Also, the AFP gene is expressed in most hepatomas. 2. Using gel retardation (band-shift) assays under very stringent conditions we have compared the binding of trans-acting factors to the proximal enhancer (-202, +34) region of the AFP gene. 3. We have detected the presence of two retarded bands in experiments performed with adult rat hepatocytes and the Fa32 cell line (which does not produce AFP) but only one band is observed with the HepG2 cell line (which produces AFP) and fetal liver. 4. We relate the two retarded bands to a glucocorticoid response element and, tentatively, to the C/EBP trans-acting fractor.


Nitric oxide is not involved in the initiation of insulin secretion from rat islets of Langerhans. The involvement of nitric oxide as an intracellular messenger in the control of insulin secretion from pancreatic Beta cells was studied in rat islets of Langerhans by measuring: (i) nitric oxide generation in response to physiological insulin secretagogues; (ii) the effects of inhibitors of nitric oxide synthesis on insulin secretory responses to physiological secretagogues, and on insulin synthesis; (iii) changes in islet cyclic guanosine monophosphate in response to secretagogues; (iv) the effects of exogenous cyclic guanosine monophosphate and dibutyryl cyclic guanosine monophosphate on insulin secretion from electrically permeabilised islets and from intact, respectively. These studies produced no evidence that nitric oxide generation is required for the initiation of insulin secretion by common secretagogues. However, the results of our experiments suggest that the generation of nitric oxide may be involved in long-term, glucose-dependent increases in cyclic guanosine monophosphate content of islet cells, although the physiological relevance of these changes requires further investigation.


Expression of c-kit, a proto-oncogene of the murine W locus, in cerebella of normal and neurological mutant mice: immunohistochemical and in situ hybridization analysis. The c-kit proto-oncogene encodes a receptor tyrosine kinase and is allelic with the murine white-spoting (W) locus. Although no apparent defects in the brain have been reported in W mutant mice, brain tissue, especially cerebellum, shows a high level of c-kit transcription. In the present study, sites of c-kit expression in the cerebellum were exained by immunohistochemical and in situ hybridization techniques. Immunohistochemistry with a monoclonal antibody against c-Kit protein revealed that the c-Kit protein was localized close to the Purkinje cell soma in the region facing the granular cell layer. Similar distribution of the c-Kit protein was observed in cerebella of mutant mice in which the Purkinje cell (pcd) or the granular cell layer (weaver) is missing. These data suggest that the c-Kit protein is produced not by the Purkinje cell nor by the granular cell but by the cells present in the molecular layer and that the protein is then transported to the region around the Purkinje cell soma. This interpretation was supported by in situ hybridization analysis: cells containing the c-kit transcripts were found only in the molecular layer, while the granular and Purkinje cells were negative.


Suprabasal marker proteins distinguishing keratinizing squamous epithelia: cytokeratin 2 polypeptides of oral masticatory epithelium and epidermis are different. Terminal differentiation of squamous epithelia is usually characterized by the synthesis of a subset of cytokeratins (CKs) in suprabasal cell layers which become major components of the intermediate filament (IF) bundle cytoskeleton of the maturing cells. We have examined the significance, molecular nature and pattern of synthesis of the elusive human CK 2 by analyzing mRNAs from certain stratified epithelia, using in vitro translation, cDNA cloning. Northern blotting and in situ hybridization. We show that genuine polypeptides with the typical gel electrophoretic mobility of CK 2 exist but that the CK 2 present in the masticatory epithelia of hard palate and gingiva (CK 2p) differs from that found in epidermis (CK 2e) by its amino acid sequence and is encoded by a different gene. The two CKs 2 show only limited sequence homology (71% identical amino acid positions in the rod domain), and the oral CK 2p is more closely related to the corneal CK 3 (86%), as is also indicated by the cross-reaction of monoclonal antibody AE5. By in situ hybridization and immunocytochemistry, we further show that both CK 2e and CK 2p are expressed only in suprabasal cell layers of the specific epithelia where they can accumulate to represent major cytoskeletal proteins. We discuss this tissue-type specificity of CK 2 synthesis in otherwise morphologically and biochemically similar epithelia in relation to differences of IF appearance and packing in upper strata between epidermal and masticatory epithelia as well as to tissue formation and differentiation during development.


Endoscopic diagnosis of early carcinoma of the esophagus using Lugol's solution. Small esophageal lesions, particularly intraepithelial cancers, are extremely difficult to detect. We used Lugol's iodine solution with panendoscopic examination to detect the presence and spread of small squamous cell carcinomas of the esophagus. Serial histologic specimens of the surgically removed esophagus from 32 patients with Lugol's combined endoscopic diagnosis of early esophageal carcinoma were examined to determine the correlation between endoscopic and histologic findings. All of the early staged carcinomas clearly remained unstained by Lugol's solution. We believe that the application of Lugol's solution will greatly aid in instances when a suspicious mucosal lesion is noted, when the margin of the lesion is unclear, or when there is suspicion that a mucosal lesion may have been overlooked.


Cuffed esophageal prosthesis: a useful device in desperate situations in esophageal malignancy. Sixteen patients (three groups) underwent endoscopic intubation with cuffed Wilson-Cook esophageal endoprostheses. Group 1 comprised 10 patients with spontaneous esophago-respiratory fistulas due to malignancy. Six primaries were esophageal, three bronchial and one ovarian. One patient could not tolerate a cuffed tube. All other fistulas closed with intubation but two tubes displaced later. Seven patients managed a soft diet after intubation, but two liquids only. Median survival was 4 weeks (range, 0 to 9 weeks). Group 2 comprised three patients with large endoscopic instrumental tears. Two had definite perforations with extensive surgical emphysema. All had satisfactory contrast swallows the day after intubation and were started on semi-solid diets; median survival was 10 weeks (one still alive). Group 3 included three patients with life-threatening arterial bleeding from cancers of the gastric cardia. No further bleeding occurred in any of the three after intubation and two survived for extended periods (15 and 26 weeks). Cuffed tubes are invaluable in these desperate situations and are worth considering for symptomatic relief even when prognosis is short.


[Significance of the maternal levels of serum alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol in various congenital developmental defects in pregnancy trimester II]. Maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG) and unconjugated oestriol (uE3) concentrations were measured in maternal serum samples from 21 pregnancies with neural-tube defects, 4 pregnancies with ventral wall defects (VWD) and 1662 unaffected pregnancies in women. These congenital malformations were confirmed by ultrasound scanning. The mean multiplate of the median (MoM) for AFP and uE3 was significantly different from the control values in cases of open NTD (AFP median MoM = 5.95, p < 0.001, uE3 median MoM = 0.2, p < 0.001), while hCG values did not differ from those of matched controls (hCG median MoM = 0.9). The biological basis of altered levels of uE3 in pregnancies with fetal NTDs is unclear.


Glial cell localization of acidic fibroblast growth factor-like immunoreactivity in the optic nerve of young adult and aged mammals. The number of axons in the optic nerve decreases with age and this degeneration is greater in patients suffering from Alzheimer's disease. Alterations in the role of neurotrophic factors could lead to this degeneration. Acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined by indirect immunofluorescence on cryostat sections incubated with a rabbit polyclonal antiserum specific for aFGF. Staining was observed by photonic microscopy on optic nerves of Wistar rats (1- to 25-month-old), bovine animals (0.5- to 7-year-old) and normal human adults (24-, 34-, 54- and 84-year-old). In the three species studied, the results show that (1) glial cells were stained in the nuclear region and (2) aFGF-like immuno-reactivity was present over a large age span in adult subjects. Endogenous aFGF may have trophic effects on retinal ganglion cells and their axons throughout the adult life span.


21-day intravenous toxicity study with feline interferon in rats. The toxicity of recombinant feline interferon, a prospective antiviral drug for cats, was examined in a subacute study. Groups of five male and five female Wistar rats were given iv feline interferon in 20 mM-NaCl at doses of 0, 5, 15 and 50 MU/kg body weight/day for 21 consecutive days. Criteria to assess toxicity included clinical observations, ophthalmoscopy, growth, food and water intake, haematology, clinical chemistry, urinalysis, organ weights, gross examination at autopsy and microscopic examination of the liver, kidneys, spleen, adrenals, heart, mesenteric lymph nodes and thymus. No treatment-related were observed even at the highest dose level. The no-observed-adverse-effect level for feline interferon in this study was therefore 50 MU/kg body weight/day.


In vivo measurement of endothelium-dependent vasodilation with substance P in man. Endothelial cells synthesize and metabolize vasoactive substances which are involved in the regulation of vascular tone. Among these factors, the endothelium-derived nitric oxide (NO) appears to be of major importance. Many studies observed an impairment of the generation, release, or the diffusion of endothelial NO across the vascular intima in laboratory animals with various experimental diseases such as hypercholesterolemia, atherosclerosis and hypertension. In human coronary arteries obtained from explanted hearts impaired endothelium-dependent relaxations were measured in atherosclerotic segments. The hypothesis of a decreased NO mediated vasodilation in patients with coronary artery disease was further underscored by in vivo studies in man using intracoronary infusions of the endothelium-dependent vasodilator acetylcholine and quantitative coronary angiographic measurements of the diameter changes. From these observations it was assumed that endothelial dysfunction, in particular a profound inability of the coronary endothelium to relax via NO dependent mechanisms may play an important role in the pathogenesis of abnormal coronary vasomotion. However, further investigations in man reveal that the ability of the coronary endothelium of patients with coronary artery disease or vasospastic angina to produce endothelial NO is less affected as judged from the effects of acetylcholine. In recent investigations a largely preserved endothelial function could be measured in these patients when the endothelium-dependent vasodilator substance P was used as a tool for the measurement of NO dependent relaxation. Thus, endothelial dysfunction does not appear to serve as a major cause of abnormal vasoconstriction in coronary artery disease or vasospastic angina in man.


Circumdural decompression by posterior vertebrectomy for relief of cord compression due to metastatic disease of thoracic and lumbar spine. Circumdural cord decompression with total or almost total vertebrectomy in the thoracic or lumbar spine through a posterior approach can provide optimal decompression of neural tissue. This procedure combined with posterior instrumentation and immediate reconstruction of the anterior defect with polymethylmethacrylate can provide adequate stabilization. Immediate ambulation with minimal external support is possible. This single operative approach is preferred to anterior (transthoracic or retroperitoneal) decompression due to high morbidity associated with the latter. The authors' experience with this procedure in four cases is presented.


Intellectual and developmental assessment of cerebral palsy cases in Libyan city. This study was undertaken to evaluate the pattern of cerebral palsy (CP) cases seen at Children's Hospital, benghazi, Libya, in respect of age and sex distribution and developmental outcome according to the type of CP and in various diseases. 60 children both male and female with CP were examined for developmental and intellectual impairment responsible for severe learning and psychomotor disabilities and to their linguistic disorder. Diagnosis of CP was made by a detailed history, physical examination and relevant laboratory investigations. Intellectual and developmental assessment of all CP cases were done by psychometry. CPs are motor syndromes, and not diseases. They are commonly associated with sensory or cognitive defects and epilepsies. The CP children when compared with normal are significant at 0.01 level of significance IQ's and DQ's. Only 8% children with mild to borderline intellectual impairment had mild speech delay an darticulation defects, and showed learning disability. 40% children with sensory disorder had variations from moderate to borderline intellectual subnormality and 36% children with birth asphyxia had delayed milestones thought were felt to have motor problems initially.


Effect of the NEP inhibitor SCH32615 on airway responses to intravenous substance P in guinea pigs. We examined the effects of the selective neutral endopeptidase (NEP) inhibitor SCH32615 on airway responses to rapid intravenous infusions of substance P (SP) and neurokinin A (NKA) and on recovery of administered tachykinins from arterial blood in anesthetized mechanically ventilated guinea pigs. SCH32615, in doses that cause a marked increase in the magnitude of bronchoconstriction induced by infused NKA, had little effect on the changes in pulmonary conductance (GL) or dynamic compliance induced by SP. In animals in which SCH32615 (1 mg/kg) was administered in combination with the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg), the dose of SP required to decrease GL by 50% was fourfold less than in animals that received captopril alone (P < 0.005). SP measured in arterial blood withdrawn within 45 s of intravenous administration of this tachykinin was not different in control and SCH32615-treated animals, whereas captopril caused an approximately threefold increase in SP concentrations (P < 0.005). When SCH32615 and captopril were administered together, significantly more SP was recovered than when captopril or SCH32615 was administered alone (P < 0.0005). Our results are consistent with the hypothesis that both NEP and ACE contribute to the degradation of intravenously infused SP. ACE degradation of SP is sufficient to limit SP-induced bronchoconstriction even in the presence of specific NEP inhibition.


Neuropeptide Y is a vasoconstrictor in human nasal mucosa. Neuropeptide Y (NPY) is a neurotransmitter in sympathetic nerve fibers in human nasal mucosa. Like norepinephrine, NPY acts as a vasoconstrictor. An established method of nasal provocation was used to determine the effects of topically applied NPY on nasal resistance to airflow measured by anterior rhinomanometry, the protein content of nasal secretions, and the protein content of bradykinin-induced secretions. NPY (2.3 nmol) reduced the resistance to inspiratory airflow by 57 +/- 18% (P < 0.001) in 10 normal subjects and by 50 +/- 17% (P < 0.05) in 12 subjects with perennial rhinitis. In nasal provocations, NPY in doses of 0.1-10 nmol had no effect on vascular (albumin), glandular (lysozyme, glycoconjugate), or total proteins present in lavaged nasal secretions. Because the vasoconstrictor properties of NPY may only be apparent in the presence of increased vascular permeability and albumin exudation, bradykinin (BK) nasal provocation was performed. BK (500 nmol) significantly increase total protein (10- to 20-fold), albumin (10- to 30-fold), and glycoconjugate (2- to 5-fold) in lavage fluid. NPY (2.3 nmol) reduced BK-induced total protein by 59 +/- 15% (P < 0.05) and albumin by 63 +/- 17% (P < 0.02) but had no significant effect on glandular secretion. Therefore exogenous administration of NPY to the human nasal mucosa reduced nasal airflow resistance and albumin exudation without affecting submucosal gland secretion. NPY agonists may be useful for the treatment of mucosal diseases characterized by vasodilation, vascular permeability, and plasma exudation.


Novel myosin isoform in nuclear chain fibers of rat muscle spindles produced in response to endurance swimming. With the use of myosin adenosinetriphosphatase (ATPase) and immunofluorescence staining methods, the adaptive responses of intrafusal and extrafusal fibers to endurance swimming were studied in frozen sections of rat soleus (SOL) and extensor digitorum longus (EDL) muscles. Glycogen depletion confirmed muscle fatigue at the end of a standardized bout of exercise. No significant age-dependent changes in myosin isoforms were detected in any fibers. The 12-wk training increased type I fibers by 10.9% in the SOL and type IIa fibers in the EDL by 16.6%. In trained muscle sections, both staining methods identified a permuted chain fiber, expressed the same as the myosin isoform in the bag2 fiber. However, no exercise-induced change of myosin isoform profile was found in the bag1 and bag2 fibers. Myosin ATPase (and immunofluorescence) staining showed the percentage of permuted chain fibers increased from 0 to 6.7% (5.6%) after 6 wk of training and to 19.2% (14.1%) after 12 wk of training and that it was still at 6.1% (4.2%) 10 wks after training. A novel myosin isoform may thus be expressed in nuclear chain fibers by repetitive recruitment of muscle spindles.


Changes in airway reactivity to exogenous and endogenous acetylcholine and substance P after anaphylactic bronchoconstriction in anaesthetized guinea-pigs. 1. In anaesthetized, actively sensitized guinea-pigs, the anaphylactic shock induced by antigen aerosol challenge (5 s; 50 mg ml-1) was followed by increase in airway reactivity to both acetylcholine and substance P. In particular dose-response curves to acetylcholine (3-1000 micrograms kg-1 i.v.) and to substance P (5-80 micrograms kg-1 i.v.) obtained in antigen exposed animals were significantly shifted to the left of those performed in control guinea-pigs (exposed to saline aerosol). 2. The hyperreactive phenomenon after antigen aerosol was also evident when capsaicin-induced bronchoconstriction (1-4 micrograms kg-1 i.v.) was tested; the degree of hyperresponsiveness was similar to that observed with acetylcholine and substance P as agonists. 3. The frequency-response curves to vagal stimulation, either cholinergic or NANC in nature, were not significantly modified in guinea-pigs challenged with the antigen in respect to those aerosolized with saline. 4. The data obtained in the present study indicate that the airway hyperresponsiveness present in the animal model used is non-specific, involving both cholinergic and peptidergic effects. On the other hand, the lack of potentiation of the bronchoconstriction response to electrical stimulation might suggest that the establishment of a clear hyperreactive phenomenon is under the control of different mechanisms unrelated to increased bronchial reactivity.


Interaction of U-50,488H with calcium channel agonists and antagonists in different cardiac tissues. 1. The present study examined the interaction of U-50,488H (specific k-agonist) with diltiazem (calcium channel antagonist) or Bay K 8644 (calcium channel agonist) on isolated left and right atria of the rat. 2. The inhibitory effects of U-50,488H on left and right atria were unaffected by the k-receptor antagonist MR-2266 (10(-7) and 5 x 10(-7) M) suggesting that they were not mediated via opioid receptors. 3. The inhibitory cardiac effects induced by U-50,488H were antagonized in presence of Bay K 8644. The negative inotropic response to the maximum concentration of U-50,488H used in presence of two concentrations of Bay K 8644 (10(-9), 3 x 10(-9) M) were 19 +/- 0.9% and 9 +/- 0.1% reductions in contractility respectively. These values were significantly (P < 0.001) lower than that obtained with the k-agonist alone (76 +/- 3.6%). Similar results were obtained for the negative chronotropism of right atria. 4. The inhibitory effect of U-50,488H was potentiating in the presence of diltiazem (5 x 10(-8) or 10(-7) M). The IC50 values for U-50,488H obtained in the left (22 +/- 2 x 10(-6) M) and right atria (610 +/- 40 x 10(-6) M) were significantly (P < 0.001) decreased in the presence of diltiazem. 5. These data demonstrate that transmembrane calcium influx may play an important role in the inhibitory cardiac effects of U-50,488H, which may be independent of k-receptor stimulation.


Anomalous reversed-phase high-performance liquid chromatographic behavior of synthetic peptides related to antigenic helper T cell sites. Sets of overlapping synthetic peptides for three well characterized proteins (sperm whale myoglobin, hen egg lysozyme, and the circumsporozoite protein from Plasmodium falciparum) were prepared and examined by reversed-phase high-performance liquid chromatography (RP-HPLC). Using retention coefficients to predict the retention time of each peptide, several peptides in each protein set were found that exhibited anomalous behavior (i.e. eluted significantly later than predicted). Previous work with model peptides has shown that this anomalous behavior can be attributed to specific amphipathic arrangements induced by the lipid stationary phase during the RP-HPLC process. In the current study it was found that although not all of the peptides containing an antigenic T cell site displayed anomalously late behavior, all of the peptides which eluted anomalously late during RP-HPLC included the regions of these proteins known from earlier studies to be antigenic T cell sites.


A comparison between one first generation and three second generation anti-HCV ELISAs: an investigation in high- and low-risk subjects in correlation with recombinant immunoblot assay and polymerase chain reaction. One first generation assay (manufactured by Ortho, test I) and 3 second generation anti-HCV ELISAs (manufactured by Ortho, Abbott, and UBI, tests II-IV) were compared. Sera from 4 different sources were used: (1) intravenous drug-users (IVDUs, n = 50), (2) blood donors (n = 1055), (3) all clinical samples from one day of routine anti-HCV testing (n = 89), (4) hemodialysis patients previously found negative by test I but clinically suspected to have a HCV infection (n = 11). Confirmatory anti-HCV tests were carried out with a second generation recombinant immunoblot assay (RIBA II). In sera positive exclusively by test IV, one antibody consumption test (UBI HCV Neutralization EIA) and one further immunoblot assay (INNO-LIA HCV Ab) were used. PCR for HCV RNA was carried out on all hemodialysis patient sera and in the RIBA II positive blood donor sera. The second generation ELISAs discriminated 11 more positive samples than the first generation test (2 IVDUs, 5 blood donors, 4 clinical samples). The 9 sera from blood donors and clinical samples were all RIBA II positive or indeterminate. The second generation tests thus showed increased sensitivity. The second generation tests also showed increased specificity in that 4 samples that were positive by test I but negative by the second generation tests, were also negative by RIBA II. With few exceptions, all RIBA II-positive and most of the indeterminate samples were positive by the second generation ELISAs. With few exceptions, all the RIBA II-negative samples were negative by the second generation ELISAs. Eleven blood donor sera were positive by test IV exclusively where RIBA II and other supplementary assays were negative. The recently introduced second generation anti-HCV ELISAs were found to have a higher sensitivity than the first generation test. The tests also showed a good concordance with the exception of test IV in the group of blood donor sera.


A quantitative reverse transcriptase-polymerase chain reaction for HIV-1-specific RNA species. The ability to evaluate the patterns and levels of human immunodeficiency virus type I (HIV-1)-specific RNA in latently and productively-infected cell lines, and primary human cells, is critical to the understanding of HIV-1 expression in cell cultures and possibly in vivo. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing in vitro transcribed RNA standards, to evaluate the copy number per cell and per microgram of total cellular RNA of multiply-spliced, unspliced and total HIV-1-specific RNA species. The latently-infected monocytic and T-lymphocyte cell lines, U1 and ACH-2 respectively, are shown to express between 10(4) to 10(6) copies of total HIV-1-specific RNA per cell, based on the state of cellular stimulation. A dramatic increase of unspliced HIV-1-specific RNA in both the U1 cell line and the ACH-2 cell line is demonstrated by this quantitative RT-PCR, 24 h after stimulation with phorbol esters. These data suggest that a single integrated HIV-1 provirus can rapidly express large quantities of HIV-1-specific RNA. Quantitative RT-PCR, for HIV-1-specific transcripts, should prove extremely useful in evaluating retroviral load and pathogenesis in cell cultures and in vivo.


A new method for measuring reverse transcriptase activity by ELISA. A new and sensitive assay of reverse transcriptase (RT) activity of retroviruses measures the incorporation of digoxigenin-labelled dUTP in newly synthesized DNA instead of radioactively labelled (3H- or 32P-)dTTP. To avoid difficulties associated with separation of non-incorporated nucleotides from the newly synthesized DNA, biotin-labelled dUTP is added to the reaction mixture in very low concentrations. After reverse transcription, the newly synthesized, doubly labelled DNA is immobilized on streptavidin-coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies (sheep) and subsequent colour development with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate(6)] (ABTSR) as substrate. For better standardization, it is suggested that RT activity is given in units (one unit of RT is the amount of enzyme incorporating one nanomole of labelled dNTP in 10 min at 37 degrees C into an acid precipitable DNA) rather than in cpm (counts per minute). The method is specific and easy to perform.


Trichogerminoma. Report of 14 cases.

Trichogerminoma. Report of 14 cases. We report 14 cases of trichogerminoma, a rare form of cutaneous adnexal neoplasm, derived from hair germ epithelium. The neoplasms occurred in 9 men and 5 women. Their ages ranged from 16 to 73 years (median 53 years). The tumors were slow growing, asymptomatic dermal or subcutaneous nodules, located on the head and neck (6), trunk (4), extremities (2) and hip (1), with no distinguishing clinical features. Histologically, trichogerminomas were characterized by sharply circumscribed, pseudoencapsulated dermal and subcutaneous nodules, ranging in size from 0.4 to 4.0 cm in diameter (mean 1.9 cm). The nodules were subdivided into lobules separated by variable amounts of stroma that demonstrated varying cellularity and mucin content. The lobules were composed of basaloid cells that formed densely packed, round nests or "cell balls" resembling hair bulbs. The basaloid cells demonstrated peripheral palisading, keratinization and differentiation towards various pilosebaceous structures. Retraction spaces, well developed hair follicles and hair shafts were not observed. These distinctive histologic features separated these neoplasms from other tumors of pilar origin and from basal cell carcinoma. The trichogerminomas behaved in a benign fashion with one exception. Complete excision of the lesions is the treatment of choice.


Mast cell quantitation by image analysis in adult mastocytosis and inflammatory skin disorders. Mast cell numbers were quantitated in adult cases of mastocytosis demonstrating non-diffuse perivascular and upper dermal concentrations of mast cells. Using the Leder stain and computerised video image analysis, a mean of 382 (+/- 28 SE) mast cell per mm2 were counted in the superficial dermis in skin biopsies from 30 adult cases of mastocytosis, in contrast to a mean of 43 (+/- 5 SE) mast cells per mm2 in skin biopsies from 50 inflammatory dermatoses represented by subacute dermatitis, pigmented purpuric dermatosis, erythema multiforme, lichen planus and granuloma annulare. Ten skin biopsies showing no significant inflammation had a mean of 54 (+/- 7 SE) mast cells per mm2 in the upper dermis. The mean area of individual mast cells as assessed by image analysis in the mastocytosis group was 47.40 microns 2 (+/- 2.26 microns 2, SE) which was significantly different (P < 0.01) than the mast cell area (32.34 microns 2 +/- 2.22 microns 2, SE) in all other groups combined. Computerised video image analysis represents an alternative technique which is useful in assessing mast cell numbers and particularly mast cell size in adult cases of macular mastocytosis and in other dermatoses.


Oral submucosal dendrocytes: factor XIIIa+ and CD34+ dendritic cell populations in normal tissue and fibrovascular lesions. Factor XIIIa+ and CD34+ dendritic cells, believed to be subsets of monocyte/macrophages, have been identified in dermis and in dermal tumors. The purpose of this study was to determine the presence and distribution of analogous cell types in oral submucosa and oral fibro-vascular lesions. Antibodies to XIIIa, CD34, S-100 protein, and macrophage antigen (MAC 387) were tested on formalin-fixed, paraffin-embedded tissue sections from normal mucosa, peripheral fibroma (PF), peripheral ossifying fibroma (POF), peripheral giant cell granuloma (PGCG), pyogenic granuloma (PG), lymphangioma (La), benign fibrous histiocytoma (BFH), idiopathic histiocytosis (IH), angiofibroma (Af) using an ABC immunoperoxidase technique. Numbers of positively stained cells were compared to unstained cells in the tumors. XIIIa positive submucosal dendrocytes (CD34-, S-100-, MAC 387-) were found in abundance in normal tissue in characteristic distributions: collagen-associated, vessel-associated, and lymphoid-associated. The percentage of XIIIa+ cells in the oral tumors was as follows: PF: 10-30%, POF: 5-10%, PGCG: 0-5%, PG: 5-20%, La: 0%, BFH: 5-25%, IH: 0%, and Af: 10-20%. CD34+ dendrocytes (XIIIa-, S-100-, MAC 387-) were few in number and were found in deeper submucosa, especially around skeletal muscle. Other than blood vascular endothelium, CD34+ cells were not generally seen in the oral tumors studied. It is concluded that two previously unrecognized dendrocyte populations reside in normal submucosa. XIIIa+ cells participate in the formation of some oral reactive and neoplastic lesions.


Monoclonal antibody analysis of keratin expression in carcinomas of sweat glands. Characteristics of keratins of five carcinomas of sweat gland origin were immunohistochemically investigated with several antikeratin monoclonal antibodies with differing specificities. Specimens were obtained from two cases of mucinous carcinoma of the skin, two cases of classic type of eccrine adenocarcinoma, and a case of eccrine porocarcinoma. The tumor cells of mucinous carcinoma expressed only simple epithelial keratins. In a case of eccrine adenocarcinoma, simple epithelial keratin 19 was diffusely expressed. The expression of the other simple epithelial keratins was confined to the luminal cells, whereas the remaining tumor cells further expressed stratified epithelial keratins. Eccrine porocarcinoma and a second case of eccrine adenocarcinoma did not express simple epithelial keratins, although stratified epithelial keratins were diffusely expressed. These data suggest that carcinomas of sweat glands express various combinations of simple and stratified epithelial keratins. Development of additional data along these lines may help to further define their classification.


Simple epithelial cytokeratin-expression in seborrheic keratosis. The cytokeratin expression of seborrheic keratosis was studied by means of immunohistochemistry and compared with that of normal human skin. The following findings were obtained in seborrheic keratosis: (1) a partial lack of high molecular weight cytokeratin (#1/68 kD, #10/56.6 kD) in all ten cases examined; (2) the detection of cytokeratin typical for simple epithelia (#8/52.5 kD, #18/45 kD, #19/40 kD) in eight of ten cases; and (3) the detection of cytokeratin #5/58 kD in suprabasal cells in 5 of 10 cases. An immunoelectron-microscopic investigation, using an anti-keratin antibody against cytokeratin #19/40 kD, revealed a whirl-like arrangement of keratin filaments within immunoreactive cells, in contrast to a linear, parallel arrangement in non-immunoreactive cells. Cells known to express cytokeratin typical for simple epithelia, such as sweat gland cells or Merkel cells, were not observed. The altered cytokeratin gene-expression in seborrheic keratosis may be attributable to de-differentiation of tumor cells or potential re-differentiation towards embryonic keratinocytes.


Removal of monocytes from cell suspensions with anti-CD14 antibody and carbonyl-iron, using Fc gamma R-dependent accessory function as a sensitive measure of monocyte presence. Human (Fc gamma RI-positive) monocytes are required as accessory cells when T cell proliferation is induced by murine IgG2a anti-CD3 monoclonal antibodies (mAbs). This T cell proliferation assay provides a sensitive method for detecting the presence of monocytes (less than 1% of monocytes can be detected), and we have used it to monitor the effectiveness of different procedures for the removal of monocytes from peripheral blood mononuclear cells. Counterflow centrifugation, phagocytosis of carbonyl-iron, adherence to plastic, monocyte depletion with magnetic beads (Dynabeads M450), and panning with anti-CD14 antibodies each strongly reduced the number of monocytes. However, none of these methods, when used on their own, were capable of completely abolishing the mitogenic response to murine IgG2a anti-CD3 mAb. A virtually complete depletion of monocytes was obtained when the panning procedure using anti-CD14 antibodies was combined with phagocytosis of carbonyl-iron. Importantly, this method could also be used with cryopreserved cells. We have applied this improved method for the removal of monocytes, to study T cell proliferation induced by murine IgG2b anti-CD3 mAb. We were able to demonstrate with this model that cells other than monocytes were able to provide accessory function.


Immunodetection of recombinant proteins based on antibodies directed against a metal binding peptide engineered for purification by immobilized metal affinity chromatography. Recently we reported (Evans, D.B., Tarpley W.G. and Sharma, S.K. (1991) Expression and characterization of chimeric rDNA proteins engineered for purification and cleavage. Protein Expr. Purif. 2, 205-213) a genetically engineered metal binding peptide (mbp) for the purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). Therefore, we have been interested in developing mbp-based immunodetection methods for these engineered proteins. To this end, the following linker peptide containing the mbp (His-Asp-His-Asp-His) was designed, synthesized and conjugated to porcine thyroglobulin: Ac-Cys-Gly-Glu-Glu-His-Asp-His-Asp-His-Pro-Phe-His-Leu. Rabbits immunized with this conjugate developed antibodies that cross-react with peptides containing the mbp sequence. A number of chimeric recombinant proteins, expressed in E. coli, with and without the mbp portion (His-Asp-His-Asp-His) of the fusion peptide (His-Asp-His-Asp-His-Pro-Phe-His-Leu) were analyzed by ELISA and immunoblotting. Results from these studies show that the anti-mbp antibodies detect chimeric proteins containing the mbp, while chimeric proteins lacking this pentapeptide were negative in both immunodetection techniques. The usefulness of this approach has also been demonstrated in following IMAC purification and enzymatic cleavage of the mbp. These immunodetection techniques utilizing anti-mbp antibodies should be applicable to other proteins engineered to contain the mbp for IMAC purification.


Recognition of Mycobacterium leprae recombinant 18-kDa proteins in leprosy. Three different, purified, Escherichia coli-derived, recombinant preparations of the Mycobacterium leprae 18K protein were compared for their immunological recognition in leprosy. The preparations tested were 18K fusion proteins containing 70% (amino acids 38-148) of the full 18K protein fused to either a short leader sequence containing six asparagine residues or to beta-galactosidase, and the full length 18K protein. All three recombinant antigens were recognized by IgG antibodies which were restricted mostly to lepromatous leprosy patients. The 18K antigen with the asparagine leader sequence showed better reactivity with IgG antibodies compared with the other two 18K preparations. In lymphocyte proliferation assays, the truncated 18K and the full-length 18K showed equivalent responses in the same donors with strongest recognition in donors who were also strongly responsive to the M. leprae soluble sonicate. These results indicate that the major human B- and T-cell epitopes are located within the segment 38-148, although some individuals may recognize additional epitopes at the NH2-terminal end.


Comparison of bis-di-octadecylamide of trehalose dicarboxylic acid (BDA.TDA) with glycolipid SL-IV as ELISA antigens for the serodiagnosis of leprosy. Two glycolipids--one synthetic and non-natural (BDA.TDA), the other natural and Mycobacterium tuberculosis species-specific (SL-IV)--were tested to determine their serological activity in sera obtained from leprosy patients, and to determine their discriminating ability in the detection of disease. The ELISA results obtained in the IgG antibody class show that both were useful substances capable of detecting multibacillary and paucibacillary disease in about 2 out of 3 leprosy patients. When these antigens were tested in parallel, the sensitivity of the ELISA test was increased by 10% without a decrease in specificity.


Enzyme linked immunosorbent assay of a 12-kDa protein of Mycobacterium leprae with sera from leprosy patients. A low molecular weight protein was obtained from a sonicate of armadillo-derived Mycobacterium leprae cells and from a lambda gt11 phage lysate of Escherichia coli (specifying the M. leprae 12-kDa protein) by a single step of ultrafiltration. Both proteins had an approximate molecular weight of about 12,000 (by SDS-PAGE) and were recognized by the M. leprae 12-kDa-specific monoclonal antibody ML06 by immunoblotting. Sera from 79 leprosy patients across the clinical spectrum, 17 contacts, and 12 normal healthy individuals were screened in an enzyme-linked immunosorbent assay (ELISA) using the 12-kDa proteins as the antigens. Antibodies to the 12-kDa protein (from lysate as well as sonicate) were detected in patients' sera across the clinical spectrum (44%-100% positivity), while no detectable reactivity was observed with control or contact sera. Sera from patients who had undergone a year or more of chemotherapy exhibited no reactivity compared to those from patients with only 3-6 months of chemotherapy. The 12-kDa proteins were also recognized by rabbit hyper-immune M. leprae antiserum.


Analysis of circulating immune complexes from leprosy patients for Mycobacterium leprae antigens. Circulating immune complexes (CICs) from 31 leprosy patients (16 tuberculoid, 15 lepromatous) and 12 healthy volunteers, precipitated by 3.5% polyethylene glycol, were individually subjected to SDS-PAGE and immunoblotting using a variety of monoclonal and polyclonal antibodies against Mycobacterium leprae. A common mycobacterial antigen of an apparent molecular size of 65 kDa was identified in CICs from about 40% of the patients. No correlation was observed between the positivity for this antigen and any of the following parameters: bacterial index, M. leprae-specific antibody titers, motor nerve involvement, duration of disease or treatment. Nevertheless, patients with a relatively recent and massive infection were more frequently positive for antigen than the others.


Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders. Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.


Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem. Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.


Inhibition of heart calcium and chloride currents by sodium iodide. Specific attenuation in cAMP-dependent protein kinase-mediated regulation. The enzymes cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate the activity of cardiac ion channel proteins. In this study the whole-cell arrangement of the patch clamp technique was used to examine the effect of NaI on PKA-stimulated Cl- and Ca2+ channels in isolated guinea pig ventricular myocytes. Cl- currents (ICl) activated either by the beta-adrenergic agonist isoproterenol or the membrane-soluble cAMP analogue, 8-chlorphenylthio (8-CPT) cAMP, were greatly reduced in amplitude after substitution of an external solution containing 140 mM NaCl with a solution containing 140 mM NaI. This reduction was accompanied by a shift of -7 mV in the reversal potential (Erev) for ICl and could be reversed upon return to the NaCl external solution. Inhibition of ICl by NaI occurred in a concentration-dependent manner and was more pronounced for inward ICl (IC50 = 19 mM at -60 mV) than for outward ICl (IC50 = 60 mM at +60 mV). In contrast to ICl activated by PKA, ICl activated by PKC was slightly augmented in the presence of NaI and the Erev was found to shift by -15 mV. Based on these data, the relative permeability of I- to Cl- (PI/PCl) for this channel was calculated to be 1.79. NaI produced no change in the amplitude of inward calcium currents (ICa) recorded under basal conditions, but strongly inhibited ICa augmented by isoproterenol and 8-CPT cAMP, and during dialysis of cells with the catalytic subunit of PKA (CS). The in vitro incorporation of [gamma-32P]ATP into histone IIA and Kemptide, measured in the presence of PKA and cAMP, was not significantly different in assay mixtures containing salts of Cl- and I-. However, the ability of isoproterenol to augment basal ICa in whole-cell experiments was attenuated when experiments were carried out entirely in NaI external solution. Thus, the reduction in ICl and ICa observed in this study may result from a direct effect of I- on the phosphorylation/dephosphorylation of cardiac ion channel proteins or associated regulatory proteins.


Tattooing as a risk of hepatitis C virus infection. The association of hepatitis C virus (HCV) infection and tattooing was studied in 87 tattooed and 126 tattoo free healthy young men who did not engage in intravenous drug use or multiple sexual activity. Antibody against HCV (anti-HCV) was tested in serum specimens by enzyme immunoassay with C100-3, NS3, and core antigens; 11 of the 87 (12.6%) tattooed and 3 of the 126 (2.4%) tattoo free subjects were positive for anti-HCV (odds ratio = 5.9, 95% CI = 1.6-22.0). A relationship was demonstrated by an increased risk for HCV infection with an increasing number of tattooed site (P(trend) = 0.002). All but one of the 87 tattooed subjects had been infected by hepatitis B virus (HBV) and 25 were carriers of hepatitis B surface antigen (HBsAg). None of the 25 HBsAg carriers was positive for anti-HCV whereas 11 of the 62 HBsAg non-carriers had anti-HCV, suggesting a negative association between the HBsAg carriage and the long lasting anti-HCV (P = 0.02, Fisher's exact). The status of the tattooer was also an important determinant for HCV infection; the risk was higher if tattooing was done by a non-professional friend than by a professional tattooist. Tattooing, probably with improperly sterilized needles, can clearly pose an increased risk for HCV infection in Taiwan. This study indicates the need for legal standards for hygienic tattooing as part of preventive measures for the control of parenterally transmitted infections.


Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-capture polymerase chain reaction. A rapid and simple RNA-capture polymerase chain reaction assay (RCPA) for detection of hepatitis C virus (HCV) is described. The assay detects specifically the presence of (near) full length genomic RNA of HCV by capturing HCV-RNA at the 3' terminal end on magnetic beads, followed by cDNA synthesis and PCR with 5' end specific primers. Sera were obtained from 30 chimpanzees inoculated with non-A, non-B hepatitis material from various sources, 28-122 months after infection. The sera were tested for the presence of HCV-RNA by RCPA and for HCV antibodies by a Line ImmunoAssay (Inno-LIA HCV Ab). Both tests were compared and show a high degree of agreement. Screening of 30 chimpanzee sera revealed either clearing of the virus below detection level (22/30) or development of a HCV carrier state (8/30). Only 1 of 11 LIA-indeterminate samples was positive by RCPA. As the RCPA is more sensitive, it can be used to test for the presence of HCV in sera which are classified indeterminate by the LIA. The outcome of the infection seems to be independent of the nature of the inocula, suggesting that the individual immune response could determine either clearing of the virus or the development of chronic infection.


Efficacy of two temephos formulations against Chironomus salinarius (Diptera: Chironomidae) in the saltwater lagoon of Venice, Italy. Two formulations of the organophosphorus insecticide, temephos (Abathion Granulare, 1% AI granular and Tambro Compresse, 2% AI tablet) were evaluated against Chironomus salinarius midge larvae in 50 x 50 m experimental plots in the saltwater lagoon of Venice, Italy. Each formulation was applied at 0.2 and 0.4 kg AI/ha. Abathion Granulare produced 56 to 73% larval reduction at 0.2 kg AI/ha and 69 to 83% reduction at 0.4 kg AI/ha during 3 wk after treatment. Abathion Granulare lost effectiveness at 4 wk after application at both rates. Posttreatment larval reductions resulting from Tambro Compresse applications ranged from 77 to 86% for 3 wk, and 82 to 92% for 4 wk at rates of 0.2 and 0.4 kg AI/ha, respectively. The tablet formulation (Tambro Compresse) gave better control of C. salinarius (magnitude and duration) than the granular formulation (Abathion Granulare) in these evaluations.


Immunohistochemical analysis of cells attached to teflon membranes following guided tissue regeneration. A wide panel of monoclonal antibodies to various keratins together with an anti-vimentin antibody to label mesenchymal cells were used to label cells attached to expanded polytetrafluorethylene (ePTFE) membranes removed 6 weeks postoperatively from 7 patients. All membranes demonstrated variable amounts of adherent tissue, within which vimentin-positive cells were detected. In 8 of the 18 samples, no epithelial cells were detected. Seven of the 18 samples demonstrated small groups of epithelial cells on both sides of the membranes, staining predominantly as basal cells. The remaining 3 samples were more densely populated by epithelium which expressed a diverse keratin profile. Thus, the membranes were successful in establishing mesenchymal cell proliferation, as evidenced by vimentin positivity, but did not totally exclude epithelium.


HIV inactivation in a bone allograft.

HIV inactivation in a bone allograft. The use of exclusionary techniques in the procurement of donors for bone allografts greatly reduces chances for disease transmission. Furthermore, treatment of HIV with either chemical agents or strong acids will effectively inactivate the AIDS virus. These data are taken as indirect proof that the risk of obtaining AIDS from a freeze-dried bone allograft is highly remote. The purpose of this study is to obtain direct evidence that the processing of a demineralized freeze-dried bone allograft would render the allograft safe for human use. In Part I, human cortical bone was obtained from a cadaveric source and tested to be free of HIV contamination. The bone was spiked with 5.26 x 10(9) viral particles. This corresponded to 148 micrograms of total viral protein. In Part II, cortical bone was procured from a donor who died of AIDS. In both Parts I and II, the cortical bone was ground to yield particle sizes of 90 to 500 microns. Test samples were treated with a virucidal agent and demineralized in HCl. Control samples were left untreated. All samples were cocultivated with stimulated peripheral blood lymphocytes and assayed for p24 core protein, reverse transcriptase, and viral gag gene by polymerase chain reaction (PCR). In Part I, the HIV spiking experiment, untreated virus infected particulate bone was positive for HIV replication. Treated samples were negative when assayed for HIV. Bone samples in Part II, HIV infected bone, were positive by PCR. Replication of viable HIV could not be demonstrated after treatment. It was concluded that demineralization and treatment with a virucidal agent inactivates HIV in spiked and infected bone.


A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques. 1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.


Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed. 1. A general approach to the analysis of ensemble currents of ligand-gated channels is presented, with a variety of examples that include single and multiple agonist binding steps, desensitization and several blocking pathways. 2. The use of matrix methods to describe model reaction schemes leads to a simplification if the reaction scheme is irreversible: the product of the exponential relaxation rate constants is exactly equal to the product of the forward reaction steps. 3. This method of analysis applied to the bovine adrenal nicotinic receptor suggests that in the range of acetylcholine concentrations from 1 microM to 2 mM, a model with a single kinetically relevant agonist binding step is appropriate. 4. Complex models, to explain the presence of two desensitizing components in currents recorded from whole cells, may be discounted in favour of two distinct receptor types. 5. A simple model of open channel block is discounted, and desensitization of the blocked state proposed.


Ca(2+)-activated K+ channels modulate muscarinic secretion in cat chromaffin cells. 1. This study was aimed at testing the hypothesis that Ca(2+)-dependent K+ channels regulate the release of catecholamines mediated by muscarinic stimulation of cat adrenal chromaffin cells. Two parameters were measured: the secretory response to brief pulses of methacholine (100 microM for 10 s) in intact cat adrenal glands perfused at a high rate with oxygenated Krebs solution; and the changes in cytosolic Ca2+ concentrations, [Ca2+]i, produced by puff applications of methacholine pulses (also 100 microM for 10 s) in isolated single cat adrenal chromaffin cells loaded with Fura-2. 2. A pulse of methacholine released 805 +/- 164 ng of catecholamines (mean of thirty-two pulses). d-Tubocurarine (DTC) increased the secretory response in a concentration-dependent manner. The maximum increase (around 1000 ng catecholamines over control values) was reached at 100 microM-DTC and the EC50 was around 10 microM. 3. The secretory responses to methacholine alone, or to the combination of methacholine plus DTC, were strongly dependent on the extracellular Ca2+ concentration, [Ca2+]o. Thus Ca2+o removal from the perfusing solution for 5-10 min abolished catecholamine release. 4. At 0.1 microM, isradipine (an L-type Ca2+ channel blocker) inhibited by 71% the secretory response to DTC plus methacholine. At 1 microM, Bay K 8644 (an L-type Ca2+ channel activator) increased 2-fold the secretory response to DTC plus methacholine (2746 ng of catecholamines). 5. Apamin (1 microM) increased 3.5-fold the secretory response to methacholine pulses (from 500 to 1800 ng of catecholamines). 6. Methacholine pulses enhanced [Ca2+]i from the resting level of 100 nM to a peak of 1000 nM which quickly declined to basal level. DTC (100 microM) enhanced by 20% the [Ca2+]i peak and substantially prolonged its duration. 7. Apamin (1 microM) increased by 60% the [Ca2+]i peak evoked by methacholine, and delayed the initiation of decline of the [Ca2+]i peak. 8. These results are compatible with the idea that muscarinic stimulation depolarizes the cat adrenal chromaffin cell through an unidentified mechanism. Depolarization is probably counteracted by activation of Ca2+i-dependent K+ channels. Therefore, inhibition of these channels enhances depolarization and firing of action potentials which activate voltage-dependent L-type Ca2+ channels to increase further the Ca2+i signal and the secretory response. Thus Ca2+i-dependent K+ channels, probably of the small-conductance type (SK), seem to be involved in the modulation of muscarinic-evoked catecholamine release responses in cat adrenal chromaffin cells.


Simultaneous visualization of cortical barrels and horseradish peroxidase-injected layer 5b vibrissa neurones in the rat. 1. Using diaminobenzidine (DAB) as a chromagen, horseradish peroxidase-injected neurones and cytochrome oxidase-stained barrels were visualized simultaneously in the rat vibrissa cortex. Neurones were initially tested during extracellular recording for responses to whisker deflections. This was followed by intracellular injection of the soma with horseradish peroxidase (HRP) and histological processing to visualize the HRP-stained neurone in an incubation solution which contained, in addition to DAB, cytochrome C for cytochrome oxidase (CO) reaction of the barrels. 2. Recording and intracellular staining were made in layer 5b under urethane anaesthesia. CO-stained barrels were observed in layer 4. Physiologically and morphologically characterized neurones were mostly large pyramidal neurones that responded to more than one whisker and displayed transient-type responses. 3. In tangential sections, the apical dendrite of the HRP-filled neurone was followed from the soma level upward as it ascended through the barrelfield in layer 4. The cross-section of the apical dendrite was found in the periphery of the CO-stained barrel. Using the apical dendrite as a guide, the basal dendritic field of the layer 5b pyramidal neurone was aligned on the pattern of layer 4 barrels. The soma was seen to project basal dendrites in all directions, involving one or two neighbouring barrels/columns. 4. In sixteen neurones examined in tangential sections, a complete spatial tuning map constructed by measuring sensitivity of the neurone to different whiskers could be compared to the basal dendritic field in relation to the pattern of overlying layer 4 barrels. The mean receptive field size in terms of the number of effective whiskers was 5.8 whereas the mean dendritic field size in terms of the number of barrels/columns involved was 2.2. In addition to the well-documented role of intracortical connectivity in elaboration of multi-whisker receptor fields in the cortical neurones, the role played by direct inputs from multi-whisker thalamic ventrobasal neurones was discussed.


Stretch-activated non-selective cation channels in the antiluminal membrane of porcine cerebral capillaries. 1. Single stretch-activated (SA) channels have been studied in isolated brain capillary endothelial cells as well as in the antiluminal membrane of intact porcine cerebral capillaries using the patch-clamp recording technique. 2. The SA channels were found to be cation selective and permeable to Na+, K+, Ba2+ and Ca2+. 3. With monovalent cations in the patch pipette, the channels showed inward rectification in cell-attached patches with a single-channel conductance of 37 pS at negative and 24 pS at positive clamp potentials. 4. With either 70 mM-Ca2+ or Ba2+ in the patch pipette, the current-voltage relation was linear with slope conductances of 16 and 19 pS, respectively. 5. Mean channel open probability increased with increasing pressure and with depolarizing clamp potentials. 6. Cell swelling induced by hypotonic shock activated the SA channels in cell-attached experiments. 7. The SA channel may be involved in cell volume or blood flow regulation. The contribution of these channels to the regulation of cerebrospinal salt and water content, especially in brain oedema, is discussed.


Inhibition of cytokine-induced nitric oxide production by transforming growth factor-beta 1 in human smooth muscle cells. 1. Experiments were performed to investigate the effects of human recombinant interleukin-1 beta on the production of vasoactive substances by human aortic smooth muscle cells in culture. Smooth muscle cells were cultured either on microcarrier beads for bioassay experiments, or in multiwell plates for the determination of nitrite levels. 2. Cells were grown on microcarrier beads, treated with interleukin-1 beta or vehicle (control) for 24 h, and packed in a column which was perfused with oxygenated Krebs-Ringer solution in the presence of indomethacin. The activity of the perfusates was bioassayed by measuring the changes in tension of a contracted ring of Wistar rat aorta without endothelium, and by evaluating the modulation of thrombin-induced platelet aggregation. 3. Perfusates from interleukin-1 beta treated cells evoked relaxations of the contracted detector tissues, and microcarrier beads covered with treated cells inhibited thrombin-induced platelet aggregation. Superoxide dismutase enhanced these effects whereas Methylene Blue abolished them. Control cells evoke neither relaxation nor inhibition of platelet aggregation. Interleukin-1 beta induced a time- and concentration-dependent production of nitrite. Cycloheximide and nitro-L-arginine inhibited the relaxations and the production of nitrite evoked by interleukin-1 beta-treated cells. L-Arginine but not D-arginine overcame the blockade elicited by nitro-L-arginine. Transforming growth factor-beta 1 reduced the interleukin-1 beta-dependent generation of nitrite by cultured smooth muscle cells and relaxation of contracted bioassay tissues. 4. Interleukin-1 beta, transforming growth factor-beta 1, Methylene Blue and L-arginine-related compounds did not induce significant variations of tension of the detector rings. 5. These data demonstrate that the inflammatory and immunological mediator interleukin-1 can stimulate the production of a nitric oxide-like substance(s) in cultured human smooth muscle cells leading to the activation of soluble guanylate cyclase. Liberation of transforming growth factor-beta by activated platelets may inhibit these reactions.


Treatment of metastatic testicular tumours with bleomycin, etoposide, cisplatin and vincristine (BEPV). Between August 1983 and December 1988, 47 patients with metastatic testicular tumours (44 non-seminomatous, three seminomas) were treated with two to six courses of bleomycin, etoposide, cisplatin and vincristine (BEPV). Five stage I tumours were included, three because of raised tumour markers following orchidectomy, one with vascular invasion of spermatic cord vessels and the other with both these features. Forty-four patients (93.6%) are alive and disease free 12-75 months (median 39 months) after completion of BEPV. Further treatment was necessary in 12 of the survivors. Eight had residual disease excised, one of whom received radiotherapy, one additional chemotherapy and one both radiotherapy and chemotherapy. Of the remaining four, two had radiotherapy and two second line chemotherapy. Thirty-one non-seminomatous and the three seminoma patients had small volume disease and all are in complete remission. Ten of the 13 patients with bulky disease are alive. It is concluded that BEPV is a well-tolerated, effective, first line therapy for patients with metastatic testicular tumour.


On the early emergence of reverse transcription: theoretical basis and experimental evidence. Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.


A redefinition of the Asp-Asp domain of reverse transcriptases. The rules defining the Asp-Asp domain of RNA-dependent polymerases deduced by Argos (1988) were tested in a set of 53 putative reverse transcriptases (RTs) sequences. Since it was found that some of these rules are not followed by RTs coded by bacteria, group II introns, and non-LTR retrotransposons, we present here a more strict definition of the Asp-Asp domain.


[Multifactorial analysis of the pyuria after transurethral prostatectomy]. Multifactorial analysis on 395 patients revealed important factors which prolong the pyuria after transurethral prostatectomy. They were the age of the patient, anemia and leukocytosis before surgery. These are factors which relate with the defense mechanism of the patient. Local factors, such as the duration of indwelling urethral catheter, the size of the prostate or prostatic bed and preoperative infection, were not so important for prolonging the pyuria after transurethral prostatectomy. The time of the resection and weight of the prostate had an intimate relation each other, and the former was the more important factor. The use of antimicrobials probably controlled these local risk factors, thus making them unimportant in the prolongation of the pyuria after transurethral prostatectomy.


[Intralymphatic drug therapy--a pathogenetically substantiated method of treatment in peritonitis]. The method of endolymphatic drug therapy (EDT) suggested by the authors has some advantages over the method of endoarterial therapy (which has proved to be effective in clinical practice) because it is aimed not only at control of the microbial flora, but also at correction of rheologic and microcirculatory disorders, improvement of the processes of tissue respiration and metabolism, stimulation of the intestine, prevention of the damaging effect of enzymes, toxins, and poisons on the organs and tissues of the body, and at correction of disorders of the immune status with promotion of the barrier function of the lymph nodes and the lymphatic system as a whole. EDT may be recommended for wide use in the clinic in the treatment of patients with peritonitis and other serious surgical infections unresponsive to the generally applied methods of management.


[Surgical treatment of lung cancer: therapy or palliative?]. In spite of the revolutionary innovations concerning diagnostic and intensive care techniques as well as better understanding of tumor biology during the last decade, rates of resection and long-term survival in bronchogenic carcinoma could not be improved. All efforts are to be done in investigation on practicable, suitable, and supportable screening programs for early recognition of tumors in the first stages.


Molecular mechanism of action of nicorandil. Nicorandil relaxes coronary vascular smooth muscle by stimulating guanylyl cyclase and increasing cyclic GMP (cGMP) levels (as shown first in our laboratory) as well as by a second mechanism resulting in activation of K+ channels and hyperpolarization. Therefore, we studied the relative contributions of either mechanism to the overall response in bovine circular strips of coronary arteries by simultaneously measuring changes in length and in cGMP levels through radioimmunoassay. Blockade by 10 microM methylene blue of the cGMP increases in strips precontracted by 1 microM of the thromboxane A2 analogue U46619 reduced nicorandil-induced relaxation to 30-50%, and there were no significant changes in cGMP levels. Suppression of the hyperpolarizing component of nicorandil by 80.4 mM K+ or 1 microM glibenclamide in precontracted strips reduced nicorandil relaxation to 50% (K+) or shifted the dose response to the right by a factor of two (glibenclamide) without alteration of increases in cGMP. A quantitative separation of both mechanisms of action was obtained by comparing the correlation between increases in cGMP and relaxation under conditions of inhibited versus noninhibited hyperpolarization. The results indicate that cGMP contributes to the total relaxing effect of nicorandil by 30-40% at low concentrations and 80-90% at high concentrations of nicorandil. From the experiments with glibenclamide, it can be concluded that the probable mechanism by which nicorandil hyperpolarizes is opening glibenclamide-sensitive K+ channels in coronary vascular smooth muscle and that this latter effect mimics those of other K+ channel openers such as cromakalim or pinacidil.


Treatment of ischemic heart disease in the 1990s: key issues for the physician. During recent years, understanding of the basic pathology, pathophysiology, and morbid risk of ischemic heart disease has increased immensely, but a wide gulf still exists between such knowledge and its practical application to the individual patient. Although the treatment of ischemic heart disease continues to pose many dilemmas for the physician in the 1990s, the key issues are easy to define, particularly with regard to pharmacotherapy. The basic pathologic processes and their rate of development and progression, their pathophysiologic consequences, and their morbidity and mortality risks are becoming increasingly clear. Until methods are developed to reconstitute the obstructed coronary arteries, clinical attention must be directed primarily to alleviating symptoms and ECG signs of myocardial ischemia and improving the deranged hemodynamic disorder. These primary objectives are now achievable with modern anti-ischemic drugs such as nicorandil. With the availability of efficacious drugs against clinical symptoms, ECG signs, and hemodynamic associates of ischemic heart disease, clinical attention must be redirected to retardation or even regression of the primary myocardial ischemic syndrome. This therapeutic goal can be achieved only by correction of the secondary risk factors that are present in many patients with ischemic heart disease. Only by such a comprehensive approach, namely, that of treatment allied to prevention, can the gap between knowledge and its practical application to the individual patient be narrowed.(ABSTRACT TRUNCATED AT 250 WORDS)


Relaxation of human coronary artery and arteria mammaria by K(+)-channel openers. Three agents with K(+)-channel-opening activity--nicorandil, bimakalim (EMD 52692), and EMD 56431--were tested for vasorelaxation abilities in human coronary artery and human arteria mammaria. The potency orders were bimakalim = EMD 56431 >> nicorandil for relaxation in human coronary artery and bimakalim = EMD 56431 >> nicorandil in human arteria mammaria. These data demonstrate that K(+)-channel openers are effective vasorelaxant agents in human coronary artery and human arteria mammaria. Bimakalim and EMD 56431 were more potent than nicorandil, a drug that also activates guanylate cyclase.


Differential effects of nitrovasodilators, K(+)-channel openers, and nicorandil on large and small coronary arteries in conscious dogs. When administered intravenously to conscious dogs, nicorandil, nitrovasodilators (nitroglycerin and SIN-1), and the K(+)-channel opener cromakalim exhibited marked differential effects on large epicardial and small resistance coronary arteries. Nitrovasodilators markedly increased the left circumflex coronary artery diameter at doses at which coronary resistance was not significantly decreased. In contrast, both cromakalim and nicorandil dilated large and small coronary arteries in the same conditions. When coronary blood flow was held to its control value by partial inflation of a hydraulic occluder implanted around the artery, the nitroglycerin- and nicorandil-induced increases in large coronary diameter remained unchanged whereas the response to cromakalim was reduced significantly. This coronary dynamic profile indicates that both nitrovasodilators and nicorandil directly dilate large coronary vessels, whereas the effect of cromakalim on these vessels is mediated partially through a flow-dependent mechanism. This suggests that nicorandil mainly dilates large coronary arteries through its nitroglycerin-like properties, whereas its ability to dilate coronary resistance vessels depends mainly on its K(+)-channel opener-like properties.


Cardioprotective effects of nicorandil.

Cardioprotective effects of nicorandil. The effects of nicorandil, a nicotinamide nitrate with K(+)-channel-opening activity, was investigated in several models of ischemia-reperfusion injury in conscious and anesthetized dogs or isolated buffer-perfused rat hearts. In several models of reversible ischemic injury (stunned myocardium) in dogs, nicorandil resulted in an enhanced recovery of regional systolic shortening during reperfusion after a single episode of coronary artery occlusion (10-15 min). These beneficial actions of nicorandil were not shared by the nitrovasodilator sodium nitroprusside but were mimicked by the selective K(+)-channel opener EMD 52692. In a model of irreversible ischemia-reperfusion injury (i.e., 2 h of coronary occlusion followed by reperfusion) in anesthetized dogs, nicorandil produced a marked reduction of myocardial infarct size. An equihypotensive dose of the calcium antagonist nifedipine had no significant effect; however, EMD 52692 produced the same reduction in infarct size as had nicorandil. In isolated, perfused rat hearts subjected to 20 min of low-flow (1.0 ml/min) global ischemia followed by 30 min of reperfusion, nicorandil (7 microM) resulted in a significant improvement in the recovery of isovolumic left ventricular minute work during reperfusion compared with untreated hearts. Finally, the results of in vitro experiments indicated that nicorandil (10(-6) to 10(-3) M) produced a concentration-dependent inhibition of superoxide anion free radical production by human and canine neutrophils. The K(+)-channel opener EMD 52692 also inhibited superoxide production in canine neutrophils. These results indicate that nicorandil is a highly efficacious myocardial protective agent in several animal models of reversible or irreversible ischemia-reperfusion injury.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of long-term nicorandil application on coronary arteries in conscious dogs. Nicorandil acting as both nitrovasodilator and K(+)-channel opener was analyzed with regard to its dilator activity in large coronary arteries. In addition, it was tested for potential development of tolerance in six chronically instrumented conscious dogs that received a 5-day continuous i.v. infusion. Increasing dosages of nicorandil (3, 10, and 30 micrograms.kg-1.min-1) caused dose-dependent strong increases in left circumflex coronary artery diameter (1.3 +/- 0.4%, 5.3 +/- 2.2%, and 8.4 +/- 2.8% above control, respectively). However, 100 micrograms.kg-1.min-1 nicorandil produced substantial hypotension, reflex tachycardia, and reduction of the dilator response in large coronary arteries. During long-term nicorandil administration (10 micrograms.kg-1.min-1 i.v. for 5 days), the diameter of the left circumflex coronary artery was increased by 8.8 +/- 2.5%. This was accompanied by a decrease in mean arterial pressure of 13.3 +/- 6.4% and an increase in heart rate by 41.5 +/- 21.0% compared with controls. On the fifth day of continuous nicorandil infusion, dose-response relations for both nicorandil- and nitroglycerin-induced epicardial artery dilations were shifted to 3.5-fold higher doses. We conclude that nicorandil dilates large coronary arteries in dogs, long-term nicorandil administration does not cause a development of tolerance, long-term nicorandil administration is not associated with the development of cross-tolerance to nitroglycerin, and the small shift of the dose-response relations is considered to reflect a hemodynamic adaptation process due to long-term nicorandil exposure.


Pharmacokinetic profile of nicorandil in humans: an overview. Nicorandil is rapidly and almost completely absorbed from the gastrointestinal tract. Nicorandil is not metabolized significantly by the liver during passage through the portal system (lack of first-pass effect). Thus, it easily enters the systemic blood flow, resulting in almost complete bioavailability (75-100%). The concomitant food intake decreases the rate of absorption of the drug, resulting in a delay of peak plasma concentration, but has little or no effect on maximal plasma concentration or total amount of absorbed nicorandil. After oral administration of a 5-, 10-, 20-, or 40-mg dose, there is a linear relationship between the doses and increases of maximum plasma concentrations and area under the curve, demonstrating that the pharmacokinetics of nicorandil are linear. Steady-state plasma concentrations of nicorandil usually are reached within approximately 96-120 h after continuous dosing (20 mg b.i.d.), probably due to its distribution and metabolism patterns. On average, the Cmax then is approximately 300 ng/ml, which is achieved rapidly within 30 min after drug intake. Nicorandil is bound weakly to human albumin and other plasma proteins (approximately 25%). After oral (and i.v.) administration of the drug, the apparent volume of distribution is approximately 1.0 L/kg body weight. Nicorandil is metabolized extensively, and the major route of elimination is the kidney: Less than 2% of the dose is excreted through the biliary route. As a consequence, the parent drug is excreted poorly in urine (very low renal clearance), whereas 2-nicotinamidoethanol, a pharmacologically inactive denitrated metabolite, is the major nicorandil-related compound excreted in urine. The nicotinamide/nicotinic acid biotransformation pathway contributes to the accumulation of nicorandil and 2-nicotinamidoethanol (denitrated metabolite) during repeated dosing because of the saturable merging of nicotinamide/nicotinic acid derivatives (from the nicorandil metabolism) into the NAD/NADP endogenous pool of coenzymes. The apparent elimination half-life is short (approximately 1 h), and total body clearance is close to 1.15 L/min, which is lower than the liver blood flow. Especially during repeated dosing, a slower elimination process appears that is related to only approximately 10% of the amount of nicorandil found in plasma. Most of the nicorandil metabolites are excreted during the 24-h period after dosing, with the remainder excreted more slowly (as nicotinamide derivatives). The pharmacokinetics of nicorandil are not altered significantly in elderly persons and in patients who have chronic renal impairment or liver insufficiency. Furthermore, its disposition profile is not modified when concomitant drugs such as drug-metabolizing enzyme inducers or inhibitors are given.(ABSTRACT TRUNCATED AT 400 WORDS)


Nicorandil and cardiovascular performance in patients with coronary artery disease. To establish the cardiovascular profile of nicorandil in patients with coronary artery disease, we recently conducted three studies at our institution. In two groups of patients undergoing cardiac catheterization, the effects of 20 mg nicorandil sublingually (s.l.) on, first, left ventricular hemodynamics (n = 10) and, second, coronary vasodilatation (n = 11) were investigated. In the first group, despite a significant decrease of 12% in left ventricular systolic pressure, heart rate did not increase significantly after nicorandil. Both left ventricular end-diastolic pressure (-43%) and the time constant of early isovolumic relaxation (-11%) decreased, whereas peak Vce and Vmax increased (+19%) (all significantly). In the second group, as mean aortic pressure decreased (-13%, p < 0.05), coronary sinus blood flow did not change significantly, and calculated coronary vascular resistance tended to decrease (-10%). Myocardial oxygen consumption decreased significantly by 14%. Quantitative coronary angiography confirmed a significant increase in the mean diameter of nonstenotic coronary artery segments (+ 14%, n = 43) and, importantly, in mean obstruction diameter of stenotic segments (+ 14%, n = 7) after s.l. nicorandil. In a third continuing study, the effects of intracoronary (i.c.) nicorandil (6 micrograms/kg) and isosorbide dinitrate (2 mg) on the epicardial coronary arteries were investigated in 10 patients undergoing coronary angioplasty. In nonstenotic coronary artery segments, mean coronary diameter increased significantly after either nicorandil (+ 12%) or isosorbide dinitrate (+ 17%). In stenotic segments, however, where the increase in obstruction diameter (+ 20%) after i.c. nicorandil was significant, the 8% increase of isosorbide dinitrate was not.(ABSTRACT TRUNCATED AT 250 WORDS)


Acute hemodynamic effects of nicorandil in coronary artery disease. Nicorandil is a potent vasodilator with antianginal and anti-ischemic properties that acts on both the coronary and the peripheral vascular bed. Because of its dual vasodilatory mechanisms mediated by an increase in cyclic GMP similar to that of nitrates and by a selective increase in the K+ conductance of the smooth muscle cell membrane, nicorandil unloads the right and left ventricles at rest and during exercise. Thus, compared with the classic nitrates, nicorandil is a more balanced vasodilator, i.e., it affects not only the venous capacitance vessels (as predominantly affected by nitrates) but also the arterial resistance vessels. In clinical pharmacologic trials, nicorandil has been administered intravenously (2 to approximately 14 mg) as well as sublingually and orally in single doses of 10-60 mg. In patients with coronary artery disease and impaired left ventricular function, the decrease in preload resulted in a significant improvement of cardiac output. Depending on the doses applied and the patient populations studied, observed decreases in systemic blood pressure were consistent with decreases in peripheral vascular resistance. The effect on heart rate was small, and no influence on atrioventricular conduction could be detected. Nicorandil enhanced exercise capacity in patients with ischemic heart disease. This benefit can be attributed to the reduction in loading of the right and left ventricle as well as to an improvement of regional wall motion abnormalities secondary to the coronary dilatory properties of the drug. Nicorandil appears to be a valuable additive to the antianginal and anti-ischemic management of coronary artery disease.


Venodilatory effects of nicorandil in healthy volunteers. The effects of a single dose of nicorandil (40 mg orally) on human dorsal hand veins in vivo were assessed and compared with the effect of nitroglycerin (0.8 mg sublingually). The hand veins had been preconstricted with norepinephrine to approximately one-third of their initial size. We demonstrated that a venous relaxation occurred after administration of nicorandil and nitroglycerin. In the doses studied, the effect was more pronounced and lasted longer after nicorandil. The pharmacodynamic effects of nicorandil in humans may be explained in part by this venous relaxation.


Efficacy of nicorandil versus propranolol in mild stable angina pectoris of effort: a long-term, double-blind, randomized study. Nicorandil is a potent coronary vasodilator. To assess its long-term antianginal effect, we designed a randomized, parallel double-blind trial of 6 weeks' duration comparing nicorandil (10 or 20 mg b.i.d.) with propranolol (40 or 80 mg t.i.d.). The study comprised 77 men with stable angina, no maintenance medication at entry, and an exercise test positive for angina and ST-segment depression. The therapy was started with 10 mg nicorandil b.i.d. or 40 mg propranolol t.i.d. After 3 weeks, the dosage could be doubled according to clinical criteria. Four men receiving nicorandil and one receiving propranolol were withdrawn with side effects; in three cases, the data were not complete. Thus, comparative data were obtained in 69 patients; in 51 of these (26 receiving nicorandil and 25 receiving propranolol), the dosage was increased to the higher level. Blood pressure and heart rate were unaltered by nicorandil and lowered by propranolol. The number of anginal attacks decreased relative to baseline on nicorandil and propranolol (p < 0.002), but total exercise duration was not influenced by either drug. The exercise test performed 2 h after either pill ingestion showed a decrease and a delay in occurrence of myocardial ischemia. The test performed 12 h after medication exhibited reduced ischemia, whereas only propranolol resulted in delayed ST-segment depression. The double product of heart rate and systolic blood pressure was affected only slightly by nicorandil and reduced significantly by propranolol (p < 0.001). Thus, nicorandil medication affords similar improvement as propranolol in patients with angina pectoris, but the mode of action appears to be different.


Antianginal and anti-ischemic efficacy of nicorandil compared with nifedipine in patients with angina pectoris and coronary heart disease: a double-blind, randomized, multicenter study. Patients with stable, effort-induced angina pectoris and a typical combination of anginal pain and ischemic ST depression in exercise tolerance tests were randomized to treatment for 8 weeks with nicorandil (a newly developed antianginal and anti-ischemic drug) or nifedipine. After 4 weeks, the dosage of nicorandil was increased from 10 mg b.i.d. to 20 mg b.i.d., but the recommended dosage of nifedipine, 20 mg b.i.d., was kept constant during the study period. Double-blind treatment was preceded by a 2-week prephase during which patients were treated with isosorbide dinitrate. During the study period, patients were asked to report the rate of anginal attacks and consumption of sublingual nitroglycerin. Measurements of blood pressure and heart rate at rest and during exercise always were performed 2 h after drug intake. Fifty-eight patients were randomized--29 to nicorandil and 29 to nifedipine. There were large individual variations in anginal attack rates, which makes group comparisons difficult, but in the nicorandil group, the anginal attack rate decreased significantly compared with baseline frequency. Systolic blood pressure at rest was reduced significantly only with the highest dose of nicorandil, but nifedipine had a significant effect on both systolic and diastolic blood pressures as well as on the heart rate. Both treatments significantly increased exercise duration, time to onset of angina pectoris, and time to 1-mm ST depression. In the nicorandil group, an improvement was noted with the 20-mg dose compared with the 10-mg dose, but no significant differences were noted between the nicorandil and nifedipine groups after either 4 or 8 weeks of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)