Antianginal and anti-ischemic efficacy of nicorandil in comparison with isosorbide-5-mononitrate and isosorbide dinitrate: results from two multicenter, double-blind, randomized studies with stable coronary heart disease patients. The purpose of the two double-blind studies summarized in this article was to compare the antianginal and anti-ischemic effects of nicorandil with those of two different nitrate preparations. A total of 129 patients with stable New York Heart Association functional class II or III coronary heart disease were enrolled in the studies. Ninety-five patients received nicorandil, 34 received isosorbide dinitrate (ISDN), and 63 received isosorbide-5-mononitrate (MN). In study 1, nicorandil was compared with MN in a crossover design with 54 protocols eligible for efficacy assessment of MN and 52 eligible for nicorandil, respectively. Twenty milligrams of nicorandil and 20 mg MN administered b.i.d. for 4 weeks were equally effective in the treatment of stress-induced angina. Both drugs prolonged bicycle exercise tolerance and reduced weekly anginal attack rates. In study 2, nicorandil and ISDN were administered to two parallel groups of patients at a dose of 10 mg t.i.d. for 2 weeks and then 20 mg t.i.d. for 4 weeks. Under the assumption that the repetitive administration of nitrates with short dosing intervals might induce the development of tolerance to the nitrate mechanism of action, the t.i.d.-dosing regimen had been chosen in this study. Thirty-two protocols from those receiving nicorandil and 34 protocols from those receiving ISDN were eligible for efficacy assessment. Both drugs increased exercise capacity and reduced ST-segment depression at identical work loads with no significant difference between groups (p > 0.05). For both drugs, the higher doses were more effective than the lower doses. tolerance to the nitrate mechanism of action did not develop with either drug.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacological characterization of nicorandil by 86Rb efflux and isometric vasorelaxation studies in vascular smooth muscle. Nicorandil and cromakalim were found to stimulate 86Rb efflux (a marker of K+ ions) from resting preparations of rabbit aorta. This action was suppressed by 10(-5) M glibenclamide, an antagonist of K(+)-channel openers in vascular smooth muscle. Through intracellular production of cyclic GMP, and subsequent suppression of cellular Ca2+ activation, nitrovasodilators interfere indirectly with the activation of Ca(2+)-dependent ion channels. 8-Bromo-cyclic GMP and sodium nitroprusside antagonized the Ca(2+)-dependent 86Rb efflux induced by 3 x 10(-7) M norepinephrine. When nicorandil and cromakalim were investigated in the same experimental setup in the presence of glibenclamide to suppress stimulation of K+ channels, only nicorandil also suppressed the norepinephrine-induced increase of the 86Rb efflux. These results confirm that nicorandil acts as both an opener of K+ channels and a nitrovasodilator. Nicorandil relaxed helical strips from rabbit aorta contracted by 10(-7) M norepinephrine, but in contrast to the relaxant action of cromakalim, this response was not antagonized by the use of glibenclamide, indicating a greater importance of the nitrovasodilator mechanism than of the K(+)-channel-opening activity for relaxation in this tissue. However, when the nitrate-like action of nicorandil was suppressed by 10(-5) M methylene blue, the K(+)-channel-opening activity was unmasked on addition of 10(-4) M glibenclamide at high concentrations of nicorandil.


Prevention of coronary spasm by nicorandil: comparison with nifedipine. The efficacy of nicorandil was compared with that of nifedipine in 13 patients with vasospastic angina enrolled in a randomized, placebo-controlled, crossover study. All patients had a coronary spasm during coronary arteriography, either spontaneously or ergometrine-induced. During two consecutive periods of 2 days, patients received active drugs or placebo in a randomized order. Each patient received single oral doses of 30 mg nicorandil, 10 mg nifedipine, and, on 2 days, a placebo. One hour after drug intake, patients underwent an ergometrine test with increasing doses of Methergin (ergometrine) (0.05, 0.10, 0.20, and 0.40 mg every 5 min). After placebo, the tests always were positive, and the ECG changes occurred at the same +/- 1 dose of ergometrine in 10 cases, showing good reproducibility. After nicorandil, the tests were negative in nine patients and positive for a higher or lower dose of ergometrine in three and one patient, respectively (p = 0.0034 vs. placebo). After nifedipine, the tests were negative in five patients and positive for a higher or the same dose of ergometrine in four and four patients, respectively (p = 0.0039 vs. placebo). Nifedipine (10 mg) and nicorandil (30 mg) were equally effective in eight patients; in the remaining five patients, nicorandil had better results (p = 0.06). Nicorandil (30 mg) prevents ergometrine-induced coronary spasm. This compound may be beneficial in patients with vasospastic angina.


Selected issues from an overview on nicorandil: tolerance, duration of action, and long-term efficacy. Nicorandil exerts its pharmacodynamic effects through two different modes of action: increase of cyclic GMP and activation of K+ channels of smooth muscle cells. The latter mechanism could explain the lack of development of tolerance that had been demonstrated clearly in various animal experiments. Therefore, it was attempted to elucidate this mechanism in humans. In 16 healthy subjects, the acute hemodynamic effects of a single oral dose of 40 mg nicorandil were compared with those of a single sublingual dose of 0.8 mg nitroglycerin. Thereafter, the subjects were treated with 30 mg isosorbide-5-mononitrate (IS-5-MN) slow release t.i.d. for 7 days to induce nitrate tolerance. Two hours after the last dose of IS-5-MN, hemodynamic measurements were repeated before and after administration of nicorandil and nitroglycerin, respectively. Recordings obtained from impedance cardiography and finger plethysmography as well as measurements of systolic time intervals provided evidence that nicorandil in the state of IS-5-MN tolerance continued to exert hemodynamic effects similar to those exerted after the first dose. The hemodynamic actions of nitroglycerin were no longer observed after chronic treatment with IS-5-MN. The duration of action of nicorandil was investigated in 22 patients with coronary artery disease (CAD). After a 2-week run-in period on placebo, patients had to perform a symptom-limited bicycle exercise tolerance test (ETT) on two separate days in which the reproducible anginal threshold had to be proven. After qualification, patients were allocated randomly to receive double-blind 10 or 20 mg nicorandil b.i.d. for 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)


Clinical profile of nicorandil: an overview of its hemodynamic properties and therapeutic efficacy. Nicorandil is a new vasodilator agent. Efficacy and safety of nicorandil in the treatment of angina pectoris have been evaluated through an extensive clinical program with a total of 1,680 patients who received the product. Results of hemodynamic studies provide clear evidence of the vasodilatory effect of nicorandil. In a population of patients with normal left ventricular function, a reduction in preload was apparent from a decrease in left ventricular end-diastolic pressure from 7.4 +/- 1.7 to -3.2 +/- 1.5 mm Hg. Furthermore, nicorandil produced marked reductions in total peripheral resistance (19%) and aortic blood pressures with decreases in systolic pressure of 34% and in diastolic pressure of 21%. At antianginal doses, nicorandil has a coronary vasodilating effect as well as a balanced peripheral action that leads to decreases in both preload and afterload. Therefore, nicorandil affects two of the main hemodynamic determinants of oxygen demand without impairing myocardial contractility or atrioventricular conduction. In addition, its strong spasmolytic activity is of particular interest when dynamic coronary obstruction is considered. Nicorandil clearly has demonstrated K(+)-channel-opening activity. In addition, the range of plasma concentrations in humans at therapeutic doses is similar to that of experimental models in which the K(+)-channel activity has been determined. This mechanism of action may explain the different hemodynamic profiles of nicorandil and nitrates in humans. Nicorandil is an effective and potent antianginal agent at a dose of 10-40 mg, which in monotherapy controls 69-80% of patients with stable chronic angina. Comparative trials have shown that the efficacy of nicorandil compares with that of drugs from the main classes of antianginal drugs--beta-blockers (atenolol, propranolol) and a Ca2+ antagonist (diltiazem). Patients treated for as long as 3 months or 1 year have shown sustained efficacy with no evidence of development of tolerance to the drug. The long duration of action allows effective treatment with a well-tolerated b.i.d. regimen. At the recommended doses, the main side effects were limited to headaches. They usually occurred early in the course of treatment and can be diminished by a progressive titration. From the large safety data base, there is no evidence that nicorandil induced exacerbation of myocardial ischemia or abrupt withdrawal syndrome. Nicorandil does not adversely affect the lipid profile or the glucose level. As an antianginal drug with a novel mechanism of action, nicorandil provides a useful alternative to existing antianginal agents in the long-term management of patients with angina pectoris.


Electromagnetic gating in ion channels.

Electromagnetic gating in ion channels. There have been many attempts to develop a theoretical explanation of the phenomena of electromagnetic field interactions with biological systems. None of the reported efforts have been entirely successful in accounting for the observed experimental results, in particular with respect to the reports of interactions between extremely low frequency (ELF) magnetic fields and biological systems at ion cyclotron resonance frequencies. The approach used in this paper starts with the Lorentz force equation, but use is made of cylindrical co-ordinates and cylindrical boundary conditions in an attempt to more closely model the walls of an ion channel. The equations of motion of an ion that result from this approach suggest that the inside shape of the channel plus the ELF magnetic fields at specific frequencies and amplitudes could act as a gate to control the movement of the ion across the cell membrane.


Self-replication of chemical systems based on recognition within a double or a triple helix: a realistic hypothesis. A scenario is proposed by which non-enzymatic self-replication of short RNA molecules could occur. The hypothesis is illustrated for the self-replication of an oligopyrimidine (Y) strand. The successful replication of Y requires a series of plausible steps. The first, experimentally feasible, step involves the template-directed polynucleotide synthesis, based on Watson-Crick base pairing, of an oligopurine (R) strand using Y as the template, and chemically activated mononucleotides as the building blocks. This step will result in the formation of an oligopyrimidine.oligopurine (YR) double helix. The second step requires the use of the double helix as the template for the synthesis of a second oligopyrimidine (Y') strand from activated pyrimidine monomers. This synthesis could be facilitated by the binding of the monopyrimidines in the major groove of the YR double helix, via Hoogsteen-type base pairing with the R strand, establishing in that sense triple helix recognition. This step, if successful, should result in the formation of a new strand, Y', that runs parallel to the oligopurine strand. Y' differs from Y in that all 3'-5' phosphodiester linkages in Y are replaced by 5'-3' linkages in Y'. The resulting triple helix (YRY') is in dynamic equilibrium with YR and free Y'. In subsequent steps, unassociated Y' directs the synthesis of the complementary oligopurine (R') strand forming a new double helix Y'R' that may direct the synthesis of an oligopyrimidine strand, Y, that is expected to be identical to the first strand that started the whole sequence. An attempt is made to generalize the above hypothesis to mixed oligonucleotides containing all four bases and identify the limitations of this hypothesis.


A method for fixation of previously fresh-frozen human adult and fetal brains that preserves histological quality and immunoreactivity. A method is described that enables fixation of previously fresh-frozen and stored adult and fetal human or animal brains. The method involves fixing during thawing under controlled, cryoprotected conditions and is compatible with good histological quality and the preservation of enzymatic activity and immunoreactivity of many neural antigens. It offers considerable advantages for the storage of large amounts of tissue from which multiple samples can be taken and processed under fixation and other conditions that can be optimized for a variety of methods, many of which may be incompatible if the whole brain is fixed in a single fixative prior to storage.


The use of texture analysis for the discrimination of Nissl substance in neurons. Structural changes induced by cordotomy in the Nissl substance of identifiable spinal motoneurons innervating the white musculature of the European eel were quantified with the use of texture features calculated from digitized images. Data were evaluated and the motoneurons classified by using multivariate analysis. The study shows that there are differences in the structural organization of the Nissl substance of motoneurons taken from control and cordotomized fishes. Distinction could only be made by using texture features, as visual examination of the motoneurons did not reveal any alteration of the studied cellular substance. Reorganization of the Nissl substance might be the result of a changed protein metabolism or a changed neuronal activity pattern consequent upon cordotomy. The method employs quick and simple techniques and could be useful in several other neurobiological studies.


Clinical potency of calcium channel blockers in asthma may be related to their inhibition of receptor-mediated phasic responses in vitro. The calcium channel blockers (CCB) have been clinically effective in exercise-induced asthma. The completeness of protection with the CCB might be related specifically to inhibition of Ca2+ influx or release. To examine this hypothesis, the rank order of potency of inhibition of the CCB, nicardipine, diltiazem and verapamil on the steady-state and kinetic parameters of the phasic and tonic responses to the muscarinic receptor agonist carbachol (10 microM) and KCl (40 mM) in the intact isolated guinea-pig trachea was determined. The Ca2+ channel agonist Bay K 8644 was also examined for its effects on intracellular Ca2+. Nicardipine abolished the KCl response at both 0.1 microM and 1 microM concentrations. The amplitude of the KCl response was inhibited equally by 1 microM diltiazem (61% inhibition) and 1 microM verapamil (68% inhibition). The rate constant of onset of the KCl response was similarly inhibited 60% by diltiazem and 66% by verapamil. Nicardipine abolished the carbachol phasic response at the 1 microM concentration. The amplitude of the phasic response was inhibited equally by 0.1 microM nicardipine (61.3% inhibition), 1 microM diltiazem (64.5% inhibition) and 1 microM verapamil (71% inhibition). The rate constant of decay of the phasic response was inhibited equally by 0.1 microM nicardipine (43% inhibition) and 1 microM diltiazem (29% inhibition). The rate constant of onset of the phasic response was unaffected by nicardipine, diltiazem and verapamil. Only 1 microM nicardipine inhibited the amplitude and rate constant of onset of the tonic response. The only effect of Bay K 8644 (1 microM) was to increase the phasic response amplitude. The CCB demonstrate a similar order of potency for inhibition of the phasic responses and clinical efficacy of the CCB in exercise-induced asthma (nicardipine > verapamil > diltiazem).(ABSTRACT TRUNCATED AT 250 WORDS)


Thalidomide and the immune system. 2. Changes in receptors on blood cells of a healthy volunteer. Thalidomide (Thd) was given in two trials (total daily dose: 5 or 8 mg Thd/kg body weight, respectively) for five and three days to a healthy male volunteer, and various receptors were analyzed on white blood cells before, during and after (up to 30 days) the treatment period. There were neither marked deviations in the absolute number of total leukocytes nor in the percentage of total lymphocytes or monocytes throughout the study period. The most pronounced changes were observed in the surface receptors on CD4 ("helper cells") cells and leukocytes bearing the CD11b (Mac 1) and other integrin and adhesion receptors. Other changes included shifts in the ratio cytotoxic cells/suppressor cells as well as a reduction of the receptor density (passage from bright to dim) in T helper cells bearing CD45RO "memory" markers. Simultaneously, the number of B cells was found to be increased as was the percentage of some adhesion receptors on CD8+ cells. Unlike in previous experiments in which Thd was administered to marmoset monkeys, no effect could be seen in cells bearing the CD2 (LFA-2) epitope.


Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Escherichia coli isolate. It has been shown that retrons, retro-elements in bacteria, produce a reverse transcriptase (RT) and multicopy single-stranded DNA (msDNA) whose 5' end is covalently linked to RNA (msdRNA) by a 2'-5' phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79-nucleotide-long single-stranded DNA with monophosphate on its 5' terminus. When the retron in strain 161 is cloned into E. coli K-12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K-12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA-DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5' side. From the analysis of the RNA-DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83-nucleotide-long DNA covalently linked to RNA. This RNA-DNA compound is further processed to the final product, the 79-nucleotide-long msDNA with a terminal 5' monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA-DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA-RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA-RNA compound is autocatalytically processed.


Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product. A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.


Regulation of the Escherichia coli uncH gene by mRNA secondary structure and translational coupling. The uncH gene is one of the most poorly-expressed genes of the proton-translocating ATPase (unc) operon of Escherichia coli. We constructed in-frame lacZ fusions to uncH and used site-directed mutagenesis to decrease the stability of the putative mRNA secondary structure in the Shine and Dalgarno region for this gene. These mutations significantly increased the expression of uncH. We also used the unc-lac fusions to show that the insertion of stop codons and a frameshift mutation in uncF, the gene preceding uncH, caused a 10-fold reduction in uncH expression. Hybridization of total cellular RNA with a lacZ-specific probe indicated that transcriptional polarity could not account for the observed decrease in gene expression. These results demonstrate that uncH expression is controlled by mRNA sequences around the translational initiation region, and is translationally coupled to uncF, even in cases where the putative mRNA secondary structure is weakened or eliminated.


The gene encoding the cellulase (Avicelase) Cel1 from Streptomyces reticuli and analysis of protein domains. Streptomyces reticuli produces an unusual cellulase (Avicelase), with an apparent molecular weight of 82 kDa, which is solely sufficient to degrade crystalline cellulose. During cultivation the processing of the Avicelase to a truncated enzyme (42 kDa) and an inactive protein (40 kDa) correlated with the occurrence of an extracellular protease. After its purification this 36 kDa protease cleaved the S. reticuli Avicelase in vitro in the same manner. Using antibodies raised against the Avicelase and its truncated form (42 kDa) and gene libraries of S. reticuli DNA in the Escherichia coli phage vectors lambda gt11 and Charon 35, the Avicelase gene (cel1) was identified. Further subcloning and DNA-sequencing revealed a G+C rich (72%) reading frame of 2238 bp encoding a protein of 746 amino acids. The transcriptional start site was mapped about 180 bp upstream from the GTG start codon. A signal sequence of 29 amino acids was identified by aligning the deduced amino acids with the characterized N-terminus of the 82 kDa Avicelase. Comparison of the N-terminal amino acids from the purified proteins with the amino acid sequence derived from the Avicelase gene revealed that the truncated enzyme (42 kDa) corresponds to the C-terminal region whereas the inactive proteolytically derived protein (40 kDa) represents the N-terminal part of the 82 kDa Avicelase. Comparisons with amino acid sequences deduced from known cellulase genes indicated the presence of three putative protein domains: (i) an N-terminal part showing significant similarity with a repeat region of endoglucanase C from Cellulomonas fimi, recently shown to be a cellulose-binding domain; (ii) an adjoining region sharing homology with the N-terminal domains with unknown function of endoglucanase A from Pseudomonas fluorescens, endoglucanase D from Clostridium thermocellum and a cellodextrinase from Butyrivibrio fibrisolvens, and (iii) a C-terminal catalytic domain belonging to cellulase family E.


Differentiation of light-dye effects in the microcirculation. Activation of photosensitive compounds has been used in the treatment of tumors and as a technique to study various microcirculatory phenomena. This technique may be accompanied by deleterious effects which may complicate interpretations of experimental results. However, the relevant physiological mechanisms that induce toxicity and the light doses needed to produce different toxic reactions have not been well defined. In the current study, the rat cremaster muscle preparation was used with in vivo fluorescent television microscopy and subsequently with electron and light microscopy to evaluate toxic reactions of light activation of fluorescein isothiocyanate. The most sensitive photoactive reactions were macromolecular leakage and platelet activation, occurring with 120 J/cm2 activation energy. Macromolecular leakage was at least partially restricted by perivenular and pericapillary pericytes and there was no morphological damage with this light dose. Since macromolecular leakage was significantly inhibited by pretreatment with diphenhydramine or Compound 48/80, it is in part due to the release of histamine from tissue mast cells. 720 J/cm2 reduced the red blood cell column in the venules by over 50% due to platelet thrombus formation, an effect that was accentuated by pretreatment with indomethacin. This suggests an inhibitory role of prostaglandins in platelet thrombus formation. In addition, 720 J/cm2 caused endothelial and smooth muscle cell swelling and ruptures, gap formation, and leukocyte and protein accumulation in the vessel walls.


Monoclonal antibodies against human alpha-fetoprotein more reactive to cell-surface alpha-fetoprotein than to free alpha-fetoprotein. This paper describes the finding of monoclonal antibody (MoAb) more reactive to cell-surface alpha-fetoprotein (AFP) than to free AFP by using a simple in vitro system. Twelve mouse MoAbs, ten IgG1, one IgG2a and one IgG2b, against human AFP from hepatocellular carcinoma were obtained by the cell fusion technique. Each hybridoma supernatant was assayed by enzyme-linked immunosorbent assay (ELISA) to solid-phase AFP. The assay results showed that two MoAbs, 67D and 80G, were most reactive to AFP. 80G had a higher affinity constant than 67D, while the both reactions were similarly difficult to inhibit by free AFP in ELISA. 67D and 80G reacted with AFP on the surface of ethanol-fixed cells from the human hepatoma cell line HuH-7 and this reaction was also difficult to inhibit by free AFP in Cell ELISA. Furthermore, Western blot analysis showed that 67D and 80G were more reactive to membrane-bound AFP than other antibodies. These findings first suggest that there could be anti-AFP MoAbs preferably binding to cell-surface AFP rather than to serum AFP.


Effects of gamma-interferon and FK506 on resting B cell proliferation of New Zealand black/white F1 mice. We examined the effects of gamma-interferon (gamma-IFN) and the new immunosuppressant FK506 on resting B cell proliferation of New Zealand black/white F1 hybrid (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE). gamma-IFN and FK506 inhibited in a dose-dependent manner both B cell proliferation and autoantibody production of resting B cells respectively. There was a synergistic interaction between gamma-IFN and FK506 in their inhibition and they did not exhibit cell cytotoxicity. This in vitro synergism of gamma-IFN and FK506 may have clinical application in that low doses of gamma-IFN and FK506 combinations may be effective to correct polyclonal B cell activation of patients with SLE.


The blood group antigen-related glycoepitopes: key structural determinants in immunogenesis and AIDS pathogenesis. This overview will focus on the functional and pathophysiological aspects of blood group antigen (BGA)-related glycodeterminants with regard to immunogenesis and AIDS pathogenesis. It has been postulated that in a broad range of histogenetically different tissues and organs, BGA-related glycoepitopes are expressed on the cell surface at definite stages of cell differentiation. These glycoepitopes are expressed during embryogenesis, organogenesis, tissue repair, regeneration, remodelling and maturation when 'sorting-out' of one homotypic cell population from a heterotypic assemblage of cells occurs (1). In this event, the BGA-related glycoepitopes, if being expressed on the cell surface, play roles of key structural determinants in cell-cell recognition, association and aggregation. This mechanism will be discussed in relation to immunogenesis with regard to antigen presentation, self-non-self discrimination, and positive and negative selection during thymic education. It is postulated that the appearance of BGA-related glycoepitopes on the cell membrane is a consequence of the association of major histocompatibility complex antigens (MHC) and peptides, with the subsequent elimination of cells carrying a high density of BGA-related glycoepitopes on their surface. After human immunodeficiency virus (HIV) glycoproteins are glycosylated by host cell glycosyltransferases, the virus may use the BGA-related glycodeterminants as ligands and/or receptors for expansion to a spectrum of target cells during AIDS development and generalization of the infection throughout the body. We will review the experimental evidence that supports the concept that HIV uses an alternative to the gp120/CD4 ligand/receptor system, and that the alternative mechanism is probably carbohydrate-mediated in nature.


Derivation of a basic mechanism of control for embryonic genes as a specific subset. The alpha-fetoprotein gene is conceived as being methylated in the zygote and according to the model is in a heterochromatic state and is therefore in a non-functional condition. Specific DNA methylase genes would produce methylases capable of alkylating enhancer regions of alpha-fetoprotein and certain proteins that would alter the heterochromatin condition. Also involved is a gene for the synthesis of a conformational-inducer protein that is proposed to be capable of blocking genic regions from reheterochromatizing. One of the pivotal events is the accumulation of S-adenosyl-L-methionine that reaches intracellular pool concentrations allowing other redundant active S-adenosyltransferase genes to become active. During embryogenesis specific conformational-inducer proteins would block genes such as the gene for albumin from reheterochromatizing while alpha-fetoprotein gene becomes heterochromatized during subsequent cell cycles. This heterochromatin is formed with embryonic type proteins sensitive to ribosylation-induced conformational changes. The increase in synthesis of alpha-fetoprotein followed by a decrease as albumin synthesis increases during embryogenesis is predicted by the scheme.


Human placental mast cells: a role in pre-eclampsia? The human placenta contains a significant amount of histamine, a potent vasoconstrictor of the placental vasculature, shown by the author to be stored within the tissues' mast cells. The proposed hypothesis suggests that placental mast cells may have an important role in normal and, or pathological processes during pregnancy. This suggestion will be applied to the confounding problem of pre-eclampsia, a complication of pregnancy characterised by hypertension, oedema and proteinuria, which is associated with increased morbidity and mortality of mother and baby. It is postulated that pre-eclampsia reflects an inflammatory-type reaction, in which mast cell-mediated events play a significant role. The mediators released upon mast cell activation, such as histamine and prostaglandins, may be involved in the vasospasm that characterises pre-eclampsia; while processes such as uptake and clearance of vasoactive mediators by mast cells may be important in normotensive pregnancies and upset in those women who develop pre-eclampsia.


Cytokine-mediated regulation of monocyte/macrophage cytotoxicity in human immunodeficiency virus-1 infection. Monocyte/macrophage-mediated tumor cytotoxicity was studied in patients infected with human immunodeficiency virus-1 (HIV-1) at various stages [Center for disease control (CDC) classification] of the disease. using the P-815 tumor cell line as target cells, the results demonstrated reduced monocyte/macrophage cytotoxicity early in HIV-1-related disease (CDCIII, P < 0.01). This cellular dysfunction sustained during the progression of the disease. Evidence could be presented that neither exogenous application of macrophage-stimulating cytokines (e.g. interferons) nor their endogenous induction in vitro restored monocyte/macrophage cytotoxicity. However, enhanced tumor necrosis factor (TNF)-alpha production, which parallels the observed reduced capacity to lyse P-815 tumor cells, might be the major source for monocyte/macrophage-mediated cell lysis. TNF-alpha-induced cytotoxicity can be inhibited by addition of anti-TNF-alpha. Other experimental models using TNF-sensitive tumor target cells may, therefore, mimic monocyte/macrophage-mediated lysis. Suppression of monocyte/macrophage cytotoxicity in later stages of HIV-1 infection (AIDS-related complex, AIDS) could partly be reverted by treatment with the cyclooxygenase blocker, indomethacin. The responsible arachidonic acid product mediating suppression was found to be prostaglandin E2, suggesting that in addition to the direct viral interference cellular dysfunction is at least in part a result of altered cytokine regulation.


Mutation analysis of genetic diseases by asymmetric-PCR SSCP and ethidium bromide staining: application to neurofibromatosis and cystic fibrosis. The Single Strand Conformation Polymorphism (SSCP) technique is widely used in mutation analysis. We have introduced several modifications to the SSCP method, which overcome the problem of incomplete denaturation or reannealing of DNA during electrophoresis. The modifications consist of asymmetrical PCR amplification of the sequence of interest, electrophoresis with a higher concentration of acrylamide, and the analysis of the DNA fragments under u.v. light. We have applied this method to the analysis of two specific diseases: neurofibromatosis type 1 (NF1) and cystic fibrosis (CF) from PCR amplified exons. Two single nucleotide changes were observed with this method.


Tandem competitive polymerase chain reaction (TC-PCR): a method for determining ratios of RNA and DNA templates. A sensitive and accurate method for determining the ratios of RNA and DNA templates by polymerase chain reaction (PCR) is presented. A common competitor containing tandemly arranged internal standards differing from the target template by the presence of different restriction enzyme sites is coamplified with the target templates under identical conditions. Products from each template and internal standard are identified by the band pattern after digestion with the restriction enzyme. As the amount of the common competitor is kept constant for all target templates, the ratio of PCR products from the templates reflects their ratio in the reaction mix before amplification. The method was used to study the relative abundance of mRNA for the pro-alpha1 and pro-alpha2 chains of type I collagen and for estimating disturbances of normal ratio in the inherited bone disorder, osteogenesis imperfecta.


A comparison of procedures for analysing microsatellite (CA)-repeat polymorphisms. Dinucleotide repeat sequences ('microsatellites') have been used as polymorphic genetic markers following amplification in the polymerase chain reaction (PCR). We have compared several methods of analysing the PCR products. The most reliable and unambiguous results were obtained when the PCR products were probed with a specific dinucleotide repeat oligonucleotide, so that only the microsatellite-containing products were detectable.


Genotoxicity of heated cooking oil vapors. Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.


Urban air pollution: use of different mutagenicity assays to evaluate environmental genetic hazard. The genotoxic activities associated with airborne particulate matter collected in Parma (northern Italy) have been determined. The airborne particle extracts were tested for mutagenicity using Salmonella frameshift (TA98) and base-substitution (TA100) tester strains with and without S9 microsomal activation and Saccharomyces cerevisiae strain D7 in order to determine the frequency of mitotic gene conversion and ilv1-92 mutant reversion in cells harvested at stationary and logarithmic growth phase. The relationship between mitochondrial DNA mutations and ageing, degenerative diseases and cancer prompted us to take into account the mitochondrial informational target, i.e., the respiratory-deficient (RD) mutants. The results obtained show a variability in the response for the different test systems during different months. The Salmonella mutagenicity trend was directly correlated with carbon monoxide, nitrogen oxides (NOx) and Pb concentration in airborne particulates and inversely correlated with temperature, whereas the mitochondrial genotoxic effect was higher during spring and late summer. These data suggest that the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested.


Mutagenic activity of the leachate of municipal solid waste landfill. Organic concentrates were recovered using XAD-2/8 resin adsorption from the leachates of municipal solid waste landfills and their mutagenic activities were tested for 8 months using the Ames Salmonella/microsome assay. Highly polluted leachates (COD and BOD > or = 40 mg/l) generally had equal or higher mutagenic activities than lightly polluted leachates (COD and BOD < 40 mg/l). But there was no clear difference in mutagenicity per amount of concentrate between the two leachates. These results suggest that the mutagenic activity of landfill leachate is decided to some degree by the organic concentration in the leachate. The mutagenic activities detected even in lightly polluted leachates were not so low as those of various kind of surface waters ever reported. It is suggested that it is important to investigate the mutagenic activity of the leachate for evaluation of the impact of landfill leachate on the environment.


Cytogenetic effects of diazepam in peripheral lymphocytes of self-poisoned persons. The frequencies of chromosomal aberrations and of micronuclei were determined in lymphocyte cultures of 25 patients who attempted suicide with diazepam, 6-12 h, 72 h and 30 days after self-poisoning. These data were compared with those of healthy controls. The frequencies of numerical aberrations showed a significant increase immediately after self-poisoning. However, this effect could not be detected on the 3rd and 30th days after self-poisoning.


Cytogenetic damage induced in human lymphocytes by sodium bisulfite. The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.


Mutagenicity testing of imidazole and related compounds. Ames tests have been performed with imidazole and its principal metabolites, hydantoin and hydantoic acid. N-Acetyl-imidazole, a potential metabolite resulting from the action of intestinal bacteria, and histamine, a structurally related compound which is widely distributed in mammalian tissues, have also been tested. Imidazole and histamine were also tested in the UDS assay in primary rat hepatocytes, while imidazole alone was tested in the M2-C3H mouse fibroblast malignant transformation assay. Imidazole gave consistently negative results in the Ames test, the UDS assay and the transformation assay. The three metabolites of imidazole, namely hydantoin, hydantoic acid and N-acetyl-imidazole, all gave negative results in the Ames test. Histamine gave no evidence of mutagenic activity in the Ames test or of genotoxicity in the UDS assay. These results indicate that imidazole and its metabolites are unlikely to present a mutagenic or carcinogenic hazard.


Acute cytogenetic effect of benzene on rat bone marrow cells in vivo and the effect of inducers or inhibitors of drug-metabolizing enzymes. Acute cytogenetic effects of benzene in LE rat bone marrow cells in vivo were studied. Chromosome aberrations (CA) induced by benzene consisted mainly of gaps and breaks. Cells with exchanges were rarely observed. The incidence of benzene-induced CA was at its maximum level 12 h after the p.o. or i.p. administration of benzene, dependent on the dose of benzene administered, and higher in male rats than in female rats. However, the sex difference was not observed in the repeated inhalation experiment. Chromosome damage was higher with the p.o. than the i.p. administration. LE rats were more sensitive than Wistar and SD rats to the clastogenic action of benzene. Phenobarbital and Sudan III are well known as inducers of drug-metabolizing enzymes. The peak percentage of benzene-induced CA in the rats pretreated with phenobarbital was observed 6 h after the benzene injection, and it occurred at a higher level than in the rats given only benzene. On the other hand, Sudan III pretreatment suppressed benzene-induced CA at all periods after the benzene injection. SKF-525A (a cytochrome P-450 inhibitor) and cyclohexene oxide (an epoxide hydrase inhibitor) pretreatment also suppressed benzene-induced CA.


Mutagenic activity in wastewater concentrates from dye plants. Wastewater concentrates from the wastewater treatment systems of three dye plants were tested for mutagenic activity in Salmonella typhimurium TA98 and Escherichia coli WP2uvrA using a fluctuation assay. Concentrates were prepared by passing samples of wastewater (5-6 or 30 litres) through two porous resins (XAD-2 and XAD-7) in series. S. typhimurium in the presence of microsomal activation proved to be the more sensitive marker of mutagenicity. Mutagenic responses were observed in concentrates from all three plants tested. The results show that mutagenic activity was particularly high in the incoming waters and increased after active, biological treatment. Physico-chemical treatment may be effective in decreasing mutagenic activity, but only if appropriately used.


Mutagenesis and comutagenesis by lead compounds. We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.


Stimulation of ACTH/cortisol by intravenously infused substance P in normal men: inhibition by sodium valproate. The effect of synthetic substance P (SP), infused intravenously in doses of 0.5, 1 or 1.5 pmol/kg-1/min-1 over 60 min on ACTH/cortisol secretion was evaluated in 7 healthy men. SP tests and a control test with normal saline were randomly performed at weekly intervals. During tests, SP infusion did not produce untoward side effects or changes in blood pressure. Plasma ACTH and cortisol levels were not modified when normal saline or the lowest dose of SP were infused, whereas they were significantly increased in a dose-dependent fashion when higher amounts of SP were administered. Further studies were performed in another 7 healthy men to test the possible influence of GABAergic neurotransmission on the ACTH/cortisol response to SP. For this purpose, subjects were tested with SP (1.5 pmol/kg-1/min-1) alone and on a different occasion with SP after pretreatment with the GABAergic agent sodium valproate (200 mg 16, 8 and 1 h before the SP test). Again, the administration of SP induced a significant increase in plasma ACTH and cortisol levels. The pretreatment with sodium valproate completely abolished both ACTH and cortisol responses to SP. These data demonstrate for the first time in humans that the systemic infusion of SP stimulates ACTH/cortisol secretion, suggesting the involvement of a GABAergic mechanism in the regulation of the action of SP.


Diurnal variation in 5-hydroxyindole-acetic acid output in the suprachiasmatic region of the Siberian hamster assessed by in vivo microdialysis: evidence for nocturnal activation of serotonin release. In vivo brain microdialysis was used to characterize the daily pattern of 5-hydroxyindole-acetic acid (5-HIAA) release in the region of the suprachiasmatic nuclei (SCN) in freely behaving male Siberian hamsters housed under 16L:8D. A marked diurnal variation in the concentration of extracellular 5-HIAA was apparent, with peak levels (147 +/- 5% of the daily mean; p < 0.05) occurring 2-3 h after lights-off. Smaller nocturnal rises in extracellular 5-HIAA were observed in the posterior hypothalamus and preoptic area (128 +/- 4 and 123 +/- 8% of the daily mean, respectively; both p < 0.05 vs. average daytime levels). Tryptophan loading increased 5-HIAA in SCN microdialysates by 44 +/- 6%, and this response was enhanced by localized perfusion with tetrodotoxin (TTX; 5 microM). Localized applications of KCl (150 mM) or veratridine (100 microM) decreased 5-HIAA by 62 +/- 5 or 49 +/- 11%, respectively. The effect of KCl was not significantly affected by specific calcium channel blockers. Perfusion with TTX markedly decreased SCN 5-HIAA during the dark phase, but had little effect during the light phase (42 +/- 8 vs. 12 +/- 5% suppression, respectively; p < 0.01). Addition of serotonin (3 microM) to the perfusate significantly stimulated 5-HIAA output. This treatment increased the release of 5-HIAA more during the dark than during the light phase (61 +/- 8 vs. 25 +/- 5%, respectively; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Protein kinase C modulates the release of [3H]5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels. The present studies examined the relationship between protein kinase C (PKC) and L-type voltage-dependent calcium channels in modulating the release of neurotransmitter from K(+)-depolarized rat spinal cord synaptosomes. Activators of PKC, such as phorbol 12-myristate 13-acetate (PMA), mezerein and oleoyl acetylglycerol produced a concentration-dependent potentiation of K(+)-induced release of [3H]5-hydroxytryptamine ([3H]5-HT). Enhanced release was dependent on the concentration of both Ca2+ and K+ in the superfusion medium. Calcium-independent release of [3H]5-HT or release induced by the Ca2+ ionophore were unaffected by PKC activators. Calcium-dependent release of [3H]5-HT, evoked by K+, was enhanced under similar conditions by the L-type Ca2+ channel agonists Bay K 8644 and (+)-SDZ 202-791. Nimodipine, an L-type Ca2+ channel antagonist, while having no independent effect on K(+)-induced release of [3H]5-HT, abolished the potentiative effects of Bay K 8644 and PMA. Similarly, the PKC inhibitors, polymyxin B and staurosporine, blocked effects of both PMA and Bay K 8644 on K(+)-stimulated release of [3H]5-HT. Neither PMA nor Bay K 8644 altered the uptake of [3H]5-HT. These results suggest that PKC-dependent mechanisms utilize calcium influx, via the L-type calcium channel, to modulate release of neurotransmitter and indicate a possible functional link between PKC and L-type voltage-dependent calcium channels in the spinal cord.


Capsaicin-like effect of (6)-shogaol on substance P-containing primary afferents of rats: a possible mechanism of its analgesic action. The effects of (6)-shogaol, a pungent component of dried ginger with a capsaicin-like chemical structure, on the release of immunoreactive substance P from the spinal dorsal horn were examined by in vitro superfusion of the dorsal-half slices of the spinal cord of the rat. (6)-Shogaol (30 microM to 1 mM) increased dose-dependently the release of immunoreactive substance P. The maximum effect of (6)-shogaol was observed at a concentration of 100 microM and less than a half of the effect of 10 microM capsaicin. The effect of (6)-shogaol (100 microM) was attenuated in slices from rats with dorsal rhizotomy and abolished by elimination of calcium ions from the perfusion medium. Pretreatment with (6)-shogaol in vitro inhibited the capsaicin-evoked release of immunoreactive substance P. On the other hand, systemic administration of (6)-shogaol (160 mg/kg) produced antinociception in rats, with a peak effect between 15 and 30 min and a smaller dose of 80 mg/kg was without effect. Treatment of rats with (6)-shogaol, at a dose of 160 mg/kg but not at 80 mg/kg, for 20 min significantly decreased release of immunoreactive substance P, evoked by capsaicin (10 microM), from the slices of cord. These data suggest that (6)-shogaol shares the sites of action with capsaicin, on the terminals of substance P-containing primary afferents, to release of the neuropeptide and inhibit the release of substance P, by subsequent stimulation of the primary afferents. The latter action of (6)-shogaol might be relevant to its analgesic effect.


Comparison in different tissue preparations of the in vitro pharmacological profile of RP 67580, a new non-peptide substance P antagonist. We describe the effects of RP 67580, a new non-peptide substance P (SP) antagonist, on tachykinin-induced contractions of guinea-pig ileum, trachea and urinary bladder, rabbit pulmonary artery and rat portal vein. All NK1 agonists tested (SP, Septide, SPOMe and [Pro9]SP) contracted guinea-pig ileum, trachea and urinary bladder (pD2 = 7.5 to 9.1), but they had no effect on rabbit pulmonary artery or rat portal vein (pD2 < 6). RP 67580 inhibited these effects: guinea-pig ileum, pA2 = 7.1 to 7.6; guinea-pig trachea and urinary bladder, pKB = 6.3 to 6.8. The difference in RP 67580 activity in these tissues might be due to the existence of subtypes of NK1 receptors. RP 67580 (1 microns) did not affect the contractions induced by the two NK2 agonists, NKA and [Lys5, MeLeu9, Nle10]NKA(4-10) (pA2 < 6), except in guinea-pig ileum (pA2 = 7.3-7.5) where these two NK2 agonists interact apparently with NK1 receptors. In the tissue preparations used, RP 67580 (1 micron) was without effect on contractions induced by the NK3 agonists: NKB and senktide. These results indicate the high selectivity for NK1 receptors of RP 67580. In all cases, similar results were obtained with another non-peptide SP antagonist, (+/-) CP-96,345. The present work provides further evidence that RP 67580 and (+/-) CP-96,345 exert in vitro a potent, selective and competitive antagonistic action on NK1 receptors and suggests the existence of at least two distinct NK1 receptor subtypes in some guinea-pig peripheral organs.


Immune cell imbalance in major depressive and panic disorders. We investigated subsets of peripheral immunologic cells in 12 drug-free patients affected by major depression according to DSM-III-R criteria, and who had recent evidence of somatic diseases. They were compared with 10 drug-free depressives, with 10 patients with panic disorder, and with 12 healthy volunteers, all without somatic disease. The immune subsets were measured by flow cytometry. The results showed that both groups of depressives had the same abnormalities in immune cells compared with the healthy volunteers or the panic disorder patients; in particular they presented a lower number of CD3+, CD8+ and HLA-DR+. The patients with panic attacks did not differ from healthy controls, except for CD4+ cells which were significantly lowered, even in comparison with the depressive groups. These data, although preliminary and in a small sample, suggest that some immune parameters may be influenced by the presence of a major psychiatric disorder.


Bilateral innervation of the musculus levator palpebrae superioris by single motoneurons in the monkey. The location of motoneurons innervating the musculus levator palpebrae superioris (LPS) was studied in the monkey (Macaca irus) by using Fast blue (FB) and propidium iodide (PI) as retrograde neuronal tracers. In each monkey, FB was injected into the LPS of one eye and PI into the LPS of the other eye. Neuronal cell bodies labeled with FB and/or PI were seen intermixed bilaterally through the entire rostrocaudal extent of the central caudal nucleus. While single-labeled (70%) cell bodies predominated, many double-labeled (30%) cell bodies were also encountered. Each of the double-labeled neurons was concluded to supply the LPS on both sides by way of axon collaterals.


Enhancement of the excitatory actions of GABA by barbiturates and benzodiazepines. Whole cell recordings from hippocampal CA1 pyramidal neurons using electrode chloride concentrations of 12-80 mM demonstrated that the effect of synaptic activation of GABAA receptors was dependent on the transmembrane chloride gradient. When the chloride reversal potential was positive to action potential threshold, GABAA receptor activation was excitatory, and anticonvulsant barbiturates and benzodiazepines enhanced this excitation. Enhancement of GABAergic excitation of interneurons may contribute to the efficacy of these drugs, while enhancement of GABAergic excitation of principal neurons may be an important mechanism of failure, such as occurs in the treatment of neonatal seizures.


Evans blue reduces macroscopic desensitization of non-NMDA receptor mediated currents and prolongs excitatory postsynaptic currents in cultured rat thalamic neurons. Fast application of L-glutamate, AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or kainate to cultured rat thalamic neurons revealed properties of non-NMDA (N-methyl-D-aspartate) receptors similar to those described in hippocampal neurons. The kinetics of non-NMDA receptor-mediated currents were altered by the addition of the dye Evans Blue (EB). Macroscopic desensitization was reduced and activation and deactivation kinetics were slowed. Delayed addition of EB, after desensitization of non-NMDA receptors, resulted in reactivation of desensitized receptors. Thus, both ion channel gating and entry into the desensitized state were affected. Evans blue also slowed the activation and the decay of glutamatergic miniature EPSCs (excitatory postsynaptic currents), demonstrating that receptor kinetics determine the time course of the synaptic response.


General anaesthetics inhibit the responses induced by glutamate receptor agonists in the mouse cortex. The effects of several general anaesthetics on the responses evoked by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) were investigated in mouse cortical wedges placed in a two compartment bath and superfused with a nominally Mg(2+)-free Krebs solution. Chloroform (3-6 mM) and halothane (1-3 mM) preferentially antagonized AMPA responses; thiopental (0.1-0.4 mM), diethyl ether (10-30 mM) and isoflurane (1-3 mM) antagonized both AMPA or NMDA responses while ketamine antagonized NMDA responses in a highly selective way. The antagonism of the excitatory amino acid responses exerted by the anaesthetics was non competitive in nature. The inhibition of excitatory amino acid receptor function may be one of the mechanisms whereby general anaesthesia is produced.


Transmitter substances contained in the petrosal ganglion cells determined by a double-labeling method in the rat. The presence of glutamate (Glu), aspartate (Asp) and substance P (SP) in the petrosal ganglion of rats anesthetized with pentobarbital sodium was studied using retrograde labeling of the carotid sinus nerve (CSN) with horseradish peroxidase (HRP) in combination with immunohistochemistry. (i) The incidence of HRP/Glu-labeled cells was the highest (32%, n = 3), followed in order by HRP/Asp-labeled cells (23%, n = 3) and HRP/SP-labeled cells (6%, n = 3). (ii) No significant difference was observed in the average diameter of HRP/Glu- and HRP/Asp-labeled cells, but the average diameter of HRP/SP-labeled cells was significantly larger than that of HRP/Glu- and HRP/Asp-labeled cells (P < 0.01). These results suggest that Glu may coexist with Asp, and SP-containing cells may form a different population from Glu- and Asp-containing cells in the petrosal ganglion. The physiological role of these transmitter substances is discussed.


Morphometric and biochemical studies in trigeminal nerve of rat after trichloroethylene or dichloroacetylene oral administration. Trigeminal nerve impairment is one of the main features of the clinical spectrum observed after trichloroethylene (Tce) exposure. A morphometric study of teased fibres, an analysis of the fatty acid composition of total lipids of the trigeminal trunk, measurements of myelin basic protein (MBP) and of 2'-3'-cyclic nucleotide 3' phosphohydrolase (CNP) activity were used to characterize this lesion. Furthermore, the effects of Tce and of dichloroacetylene (Dca), a breakdown product supposed to be the main cause of the trigeminal neuropathy, were compared under the same experimental conditions. Results showed a significant decrease of the internode length and of the mean fibre diameter in the Dca group whereas these values, although also diminished, were not significantly affected in the Tce group. In the trigeminal nerve, this was associated with a significant (46%) decrease in sigma. (n-6) fatty acid and a 22% decrease in sigma. (n-3) fatty acid contents in the Tce group whereas these decreases were respectively 32% in sigma. (n-6) fatty acid and 27% (significant) in sigma. (n-3) fatty acid contents in the Dca group. In this group, a 64% increase in sigma. Fatty aldehydes was also observed. Finally, Tce or Dca had only a slight effect on the overall profile of fatty acid content in the brain and no significant variation was observed in MBP level and CNP activity. Both compounds can thus induce trigeminal nerve alterations, but with a higher intensity for Dca.


Clinical, biochemical, electrophysiologic, and histologic assessment of chlorpyrifos induced delayed neuropathy in the cat. The ability of a supralethal dose of chlorpyrifos to produce delayed neuropathy was examined using assessments of clinical signs, electromyography (EMG), motor nerve conduction velocity (MNCV), lymphocyte neuropathy target esterase activity (LNTE), and histologic changes in nervous system tissues. Cats were exposed to a single, im injection of corn oil (vehicle control), DFP (positive control) at 5.0 mg/kg, or chlorpyrifos at 300 mg/kg and observed for 60 days. Atropine and 2-PAM were administered to chlorpyrifos exposed cats one to two times a day for 14 to 24 days in response to the appearance of cholinergic signs. Anorectic cats during the acute toxicosis were force fed by hand and hydration was maintained by administering fluids sc. Onset of ataxia (mean +/- SD) for the positive control and chlorpyrifos exposed cats were 16.2 +/- 1.8 days (range of 14-19 days) and 19.0 +/- 1.4 days (range of 17-21 days), respectively. Functional deficits for both groups were confined to the hindlimbs and characterized by a crouched-waddling gait, hypermetria, and proprioceptive deficits. Maximal inhibition of LNTE activity was 96% at 24 hr postdosing in the positive control group and 46% at 7 days postdosing in the chlorpyrifos group. No EMG or MNCV abnormalities were detected in any of the treatment groups. Axonal degeneration was similar for the positive control and chlorpyrifos exposed cats. Ascending tracts of the cervical spinal cord and descending tracts of the thoracic and lumbar spinal cord were most severely affected and peripheral nerves were only mildly affected. The clinical and histologic effects produced indicate that chlorpyrifos can cause delayed neuropathy in the domestic cat. The moderate but prolonged inhibition of LNTE produced by chlorpyrifos is atypical of classic organophosphorus delayed neurotoxicants.


Intravitreal neovascular tissue of proliferative diabetic retinopathy: an immunohistochemical study. Intravitreal neovascular tissue in 8 cases of proliferative diabetic retinopathy was investigated using immunohistochemical techniques. All 8 cases yielded positive immunoreactivity for type II collagen (vitreous collagen). The intravitreal neovascular tissue was classified into two groups (A or B), depending upon the distribution of type II collagen. In group A (3 cases), blood vessels were entirely surrounded by vitreous collagen, and in group B (5 cases), the vessels proliferated on one side of a mass of vitreous collagen. Type I and III collagens were distributed diffusely within the extracellular space of the tissue, whereas type IV collagen and fibronectin (FN) formed a basement membrane-like foundation for the newly formed vessels. Glial fibrillary acidic protein (GFAP)-immunoreactive cells were not clearly detected in any of the cases. Neovascular tissue typically proliferated on the posterior vitreous surface (as found in group B), but was also found to penetrate the vitreous gel (as found in group A). As neovascular tissue proliferation proceeded, types I, III and IV collagens and FN were produced. Glial cells (GFAP-positive cells) were not essential for neovascular tissue formation.


Mass cultivation of human retinal pigment epithelial cells with microcarrier. Microcarrier cell culture permits mass cultivation of anchorage-dependent cells. In this study, mass cultivation of human retinal pigment epithelial (RPE) cells was studied using Cytodex 3 (Pharmacia) as a microcarrier. Human RPE cells were established from aborted fetuses and cultured in Dulbecco's modified Eagle's Medium (DMEM). After the 3rd-5th passages, RPE cells were suspended in 50 ml of DMEM in a spinner flask at a density of 2 x 10(5)/ml, and Cytodex 3 was added to the spinner flask at a bead density of 10 mg/ml. Cultures were maintained at 20-50 rpm (final speed) on a magnetic stirrer, and DMEM was added up to 100 ml. Fifty milliliters of DMEM were decanted and replaced with fresh DMEM every 2 days. After 1 week, a cell density of 10(6)/ml DMEM was obtained. Phase contrast microscopy showed bridging formation between microcarriers, which suggests tight cell adhesion. Microcarrier cell culture has a variety of advantages which include greater cell production, use of less medium and less risk of contamination compared to the conventional monolayer culture technique, and it also allows passaging without using proteases. Using this culture system, greater possibilities for wider application of new cell cultures can be expected.


[Informative value of varying serum alpha fetoprotein levels in pregnant women based on a screening program in a Hungarian institute. What next?]. The authors evaluated the maternal serum alfa fetoprotein screening data of 3 years period. Out the neural tube defects they observed increased values at 20% of another congenital malformations. At the pregnant having abnormal serum alfa fetoprotein significantly accumulated the late-term abortion, stillbirth and preterm delivery. At the mothers of stillborns and abortions frequently noticed the different values. They direct attention to the fetal-living-danger importance, control functions of the abnormal level and after prospective data collection it may have new aspecific alarm role too. Accordingly the maternal serum alfa fetoprotein may consider like fetal blood sediment examination.


Identification of editing positions in the ndhB transcript from maize chloroplasts reveals sequence similarities between editing sites of chloroplasts and plant mitochondria. A comparison of the nucleotide sequences from genomic DNA and cDNA of the ndhB gene from maize chloroplasts shows that the ndhB transcript is edited by C-to-U transitions at six positions which appear to exist as editing sites also in the chloroplast ndhB genes from rice and tobacco but not from liverwort. In order to identify possible sequence determinants necessary for editing, the sequences surrounding the newly identified ndhB and previously identified ndhA editing sites were compared with each other and with editing sites observed in plant mitochondrial transcripts. Among the chloroplast editing sites two closely positioned ndhB sites show similarity by sharing a common octanucleotide. The existence of the identical octanucleotide in the ndhJ gene whose transcript is not edited at the respective position, shows, however, that this octanucleotide is not sufficient to elicit the editing process. On the other hand, several of the chloroplast editing sites show sequence similarities with certain sets of consensus sequences reported earlier for editing sites of plant mitochondria. This supports the view that the editing processes of both plant organelles share common components and/or mechanistic steps and that the consensus sequences are part of the determinants necessary for editing.


New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry. We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.


Quantitation of human cellular retinoic acid-binding protein II (CRABP-II) RNA from cultured human skin fibroblast cells and human skin biopsies treated with retinoic acid. A polymerase chain reaction (PCR) method has been validated for the quantitation of retinoic acid (RA) induction of cellular retinoic acid-binding protein II (CRABP-II) RNA from cultured human skin fibroblasts and human skin biopsies. The method utilizes reverse transcription and PCR (RT-PCR) to compare cellular CRABP-II RNA with a known amount of added internal standard RNA generated from a modified CRABP-II cDNA containing a 42 bp deletion. Thus, after RT-PCR of cellular and standard CRABP-II RNA in the same tube, the resulting DNA bands could be distinguished by size on ethidium bromide-stained, nondenaturing polyacrylamide gel. Serial dilutions of cellular RNA were co-amplified with a fixed amount of internal standard CRABP-II RNA, and the ratio of intensities of the two DNA bands was determined by computerized image analysis of the gel photograph. A linear relationship was found between the logs of this ratio and the input RNA. Absolute quantitation of cellular CRABP-II RNA was determined from the 'equivalence point', the dilution at which band intensities from cellular and standard RNAs were identical. Using this quantitative assay, the amount of CRABP-II RNA in cultured fibroblasts was 24 attomoles per microgram total RNA. A 4.2-fold increase in CRABP-II RNA was seen following 24 hours treatment with 10(-6) M RA. CRABP-II RNA content in skin biopsies taken from 3 human subjects ranged from 16 to 25 attomole/micrograms RNA. Topical treatment with 0.1% RA cream resulted in induction ranging from 3.9- to 12-fold over vehicle treatment. The method described here offers a rapid, sensitive and quantitative assay of specific RNAs, and should be especially useful for the measurement of RNA levels from small solid-tissue biopsies.


A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron. RNA polymerases can synthesize RNA containing phosphorothioate linkages in which a sulfur replaces one of the nonbridging oxygens. Only the Rp isomer is generated during transcription. A Rp phosphorothioate at the 5' splice-site of the Tetrahymena group I intron does not inhibit splicing (McSwiggen, J.A. and Cech, T.R. (1989) Science 244, 679). Transcription of mutants in which the first base of the 3' exon, U+1, was mutated to C or G, in the presence, respectively, of either cytosine or guanosine thiotriphosphate, introduced a phosphorothioate at the 3' splice-site. In both cases exon ligation was blocked. In the phosphorothioate substituted U+1G mutant, a new 3' splice-site was selected one base downstream of the correct site; despite the fact that the correct site was selected with very high fidelity in unsubstituted RNA. In contrast, the exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site. Chirality was reversed during transesterification as in 5' splice-site cleavage (vide supra). This suggests that one non-bridging oxygen is particularly crucial for both splicing reactions.


The strands of both polarities of a small circular RNA from carnation self-cleave in vitro through alternative double- and single-hammerhead structures. The sequence of a circular RNA from carnation has been determined and found to consist of 275 nucleotide residues adopting a branched secondary structure of minimum free energy. Both plus and minus strands of this RNA can form the hammerhead structures proposed to mediate the in vitro self-cleavage of a number of small infectious plant RNAs and the transcript of satellite 2 DNA from the newt. Minus full- and partial-length transcripts of the carnation circular RNA including the hammerhead structure showed self-cleavage during transcription and after purification, indicating the involvement of a single-hammerhead structure in the self-cleavage reaction. In the case of the plus transcripts only a dimeric RNA, but not a monomeric one, self-cleaved efficiently during transcription and after purification, strongly supporting the implication in this process of a double-hammerhead structure theoretically more stable than the corresponding single cleavage domain. However, a plus monomeric transcript self-cleaved after purification at a slow rate in a concentration-independent reaction which most probably occurs through an intramolecular mechanism. Comparative sequence analysis has revealed that the circular RNA from carnation shares similarities with some representative members of the viroid and viroid-like satellites RNAs from plants, suggesting that it is a new member of either these two groups of small pathogenic RNAs.


Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp. PCC 6803, and from the heterocyst-forming strains Anabaena sp. PCC 7120 and Calothrix sp. PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp. PCC 6301) as a probe. The genes and the flanking regions were sequenced. The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences. In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria. The ends of the mature RNAs were determined by primer extension and RNase protection. The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus.


Feasibility of an implantable arrhythmia monitor. Conventional Holter monitoring is of limited benefit in patients with infrequent symptoms suspected to be related to arrhythmia. A small recorder implanted subcutaneously might obviate many limitations of conventional monitoring. To determine the feasibility of obtaining adequate electrocardiographic signals from such a device, a prototype was temporarily implanted in 17 patients undergoing pacemaker implantation. The prototype contained four disc-shaped titanium electrodes, 0.21 inches in diameter embedded in epoxy. The four electrodes were in a square configuration spaced 0.72 inches center to center and were placed face down in a subcutaneous pocket in the left pectoral region. Bipolar recordings were made from a horizontal pair, a vertical pair, and both diagonal paris of electrodes (interelectrode distance 1.02 inches) and recorded on electromagnetic tape after filtering at 0.5-250 Hz. The mean peak-to-peak amplitude in each configuration was determined over a five-beat interval. Clear recordings were obtained from all 17 patients with recognizable P, QRS, and T waves. The amplitude of the signals obtained from the diagonal pairs of electrodes (175 +/- 51 and 170 +/- 54 microV) were greater than obtained from either the vertical pair (142 +/- 62 microV, P = 0.08 compared to diagonal electrodes) or the horizontal pair of electrodes (105 +/- 54 microV, P < 0.01). The maximum amplitude recorded from any configuration was 189 +/- 54 microV. In six patients the device was also tested with the electrodes face up in the subcutaneous pocket.(ABSTRACT TRUNCATED AT 250 WORDS)


Permanent pacemaker implantation in the cardiac catheterization laboratory versus the operating room: an analysis of hospital charges and complications. Permanent pacemakers may be implanted in operating rooms, special procedure laboratories, or cardiac catheterization laboratories. Previous investigators have shown no difference in efficacy or complications in the operating room versus the cardiac catheterization laboratory. We retrospectively analyzed the hospital bills of 30 patients undergoing permanent pacemaker implantation at our institution. Group I was 15 consecutive patients implanted in the operating room and group II was 15 consecutive patients implanted in the cardiac catheterization laboratory, all by the same operators. Hospital charges that were specific to the site of implantation were analyzed. Physician charges for implantation, anesthesiologist, and radiologist charges were not analyzed. There were no in-hospital complications in either group. The mean charges for group I were $1,856.00 and group II were $1,075.00 (P < 0.001). We conclude that implantation of permanent pacemakers in the cardiac catheterization laboratory is associated with significantly lower hospital charges compared to implantation in the operating room and has an equally low complication rate.


Adenosine and verapamil-sensitive ventricular tachycardia originating from the left ventricle: radiofrequency catheter ablation. A ventricular tachycardia (VT) with right bundle branch block (RBBB) QRS morphology and left axis originating from the inferoapical segment of the left ventricle is described in a 49-year-old man without structural heart disease. This VT could be initiated during isoproterenol infusion and was terminated with intravenous administration of adenosine and verapamil. Radiofrequency ablation eliminated the tachycardia. Previous reports have suggested reentry as the mechanism for a verapamil-sensitive VT with this ECG morphology, while cAMP-mediated triggered activity has been proposed as a mechanism for VTs sensitive to adenosine. The latter more typically arise in the right ventricular outflow tract. The electrophysiological and electropharmacological characteristics of the tachycardia in this patient suggest that this VT morphology is not specific for a mechanism but rather for the location of the site of origin.


New concept in activity-controlled pacemakers: clinical results with an accelerometer-based rate adaptive pacing system. An accelerometer-based rate adaptive generator (EXCEL VR) has been introduced. A preclinical group of 22 subjects with strap-on devices was observed and reported. A clinical protocol including observation of rate adaptive response to typical daily activities and incremental exercise on a treadmill was administered in seven implanted patients. Indications for implantation in these patients was either second- or third-degree atrioventricular block (five patients, VVIR pacing mode) and sick sinus syndrome (two patients, AAIR pacing mode). Mean pacing rates were 50 ppm (supine), 56 ppm (standing), 77 ppm (descending the stairs), 81 ppm (slow walk), 83 ppm (slow stair climb), 91 ppm (fast walk), and 92 ppm (fast stair climb). When the arm proximal to the pulse generator was exercised, the rate rose to 92 ppm. When the distal arm was strained, the rate was 63 ppm. During treadmill testing, rates between 82 ppm (2 km/hour) and 104 ppm (5 km/hour) were observed. This accelerometer-based rate adaptive pulse generator provided a proportional response to graded activities of treadmill exercise and daily living in these groups of preclinical and clinical subjects.


Reversible late potentials due to ischemia. The role of ischemia in the development of reversible late potentials was assessed in 19 patients undergoing percutaneous transluminal coronary angioplasty. Signal-averaged electrocardiograms were performed before angioplasty, during ischemia caused by balloon inflation and after angioplasty. Five of 19 patients developed late potentials that reverted to normal after angioplasty. Age, sex, ejection fraction, left ventricular end diastolic pressure, vessels involved, and extent of myocardium in jeopardy did not predict the development of late potentials. Patients with a prior history of myocardial infarction were more likely to develop late potentials. Therefore, patients with prior myocardial infarction appear more likely to develop the substrate for reentrant ventricular tachycardia during periods of ischemia.


Two different reentrant circuits of ventricular tachycardia in a patient with an extensive anterior infarction: evaluation using electrical catheter ablation techniques. Two morphologically distinct sustained ventricular tachycardias were initiated by programmed stimulation during attempted catheter ablation in a patient with an old anterior myocardial infarction. Right bundle branch block configuration of ventricular tachycardia, which was identical to the spontaneously occurring tachycardia, was initiated and displayed fragmented mid-diastolic potential at the apicolateral left ventricular site. Evidence of a critical slow conduction area was observed during delivery of electrical stimuli to this area. Following a 150-joule electrical shock delivered to this area, right bundle branch block pattern of ventricular tachycardia was no longer inducible but a new sustained monomorphic ventricular tachycardia with left bundle branch block pattern was initiated. The mid-diastolic fragmented activity at the ablation site became electrical activation of bystander area that was not participating in the left bundle branch block type of the ventricular tachycardia circuit. The critical slow conduction area was identified at the apicoseptal left ventricular site that was separated more than 5 cm from the ablation site. We speculate that two morphologically distinct sustained monomorphic ventricular tachycardias may be due to two different reentrant circuits and not the different expression of the same circuit.


A degenerative lesion of the approach to the atrioventricular node producing second-degree and third-degree atrioventricular block. We report a case of a degenerative approach lesion in an 83-year-old male with diabetes mellitus, hypertension, and ischemic heart disease. His ECGs changed from first-degree atrioventricular (AV) block 14 years ago, to third-degree AV (A-H) block. A pacemaker was implanted for bradycardia. He died 4 years later from heart and renal failure. Serial sections through the conduction system revealed total depletion and fatty replacement of the atrial muscle at the approaches to the AV node.


Physiology of "atypical" atrioventricular junctional reentrant tachycardia occurring following radiofrequency catheter modification of the atrioventricular node. The physiology of atypical atrioventricular junctional reentrant tachycardia (AVJRT) occurring following catheter modification of the AV node is poorly defined. Six patients undergoing radiofrequency current catheter modification of the AV node had inducible atypical AVJRT before or after AV nodal modification. Typical AVJRT was differentiated from atypical AVJRT by a ventriculoatrial (VA) time < 60 msec in the His-bundle electrogram recording. Five of six patients had typical AVJRT and two had atypical AVJRT prior to AV nodal modification. Following anterior approach AV nodal modification, previously undetected atypical AVJRT was induced in four patients. Earliest retrograde atrial activation in the posterior septum was documented in all patients with atypical AVJRT prior to modification and in three of four patients with atypical AVJRT following modification. The AH intervals during tachycardia were 320 +/- 52 msec in typical AVJRT, 88 +/- 33 msec in the premodification atypical AVJRTs, and 172 +/- 12 msec in the postmodification atypical AVJRTs (P = 0.0001). The AH/HA ratios were 4.1 +/- 0.9 in typical AVJRT, 0.5 +/- 0.2 in the premodification atypical AVJRTs, and 0.9 +/- 0.2 in the postmodification atypical AVJRTs (P = 0.0001). Two patients with postmodification atypical AVJRT underwent further posterior approach AV node modification that resulted in VA block. One patient with postmodification atypical AVJRT had further anterior approach AV nodal modification that resulted in heart block. The retrograde limb of the atypical AVJRT seen following anterior approach AV nodal modification is a posterior, slow pathway.


Detecting right ventricular volume changes using the conductance catheter. The purpose of this study was to assess the role of conductance catheter position within the right ventricle in obtaining adequate indications of phasic changes in ventricular volume. Possible applications of this technology are in rate responsive pacemakers and implantable defibrilators. The conductance catheter was placed in the right ventricle by cannulation of a jugular or femoral vein or a branch of the pulmonary artery. Position within the ventricle was documented from biplane fluoroscopy. Stroke volume was perturbed by: bolus injection of blood, vagal stimulation, venous infusion of methylcholine chloride, or isoprenaline. Four criteria were used to assess the quality of volume signals: (1) volume signal phase relative to the electrocardiogram; (2) magnitude parity of volume change from each electrode pair; (3) freedom from artifact; and (4) indication of stroke volume change during interventions. Greyhound dogs of either sex (n = 33), weight 20-32 kg. A total of 236 recordings from 14 distinct catheter positions were analyzed. Catheter positions originating from a femoral cannulation and one position from the pulmonary artery gave markedly superior volume transduction compared to those from the jugular route. Although right ventricular volume transduction was possible from all catheter trajectories, those resulting from the femoral approach were clearly superior. In the right ventricle, the inability to transduce a sufficient proportion of ventricular volume, in concert with the potential sensitivity of the catheter to atrial volume changes, may seriously limit the potential of the conductance technique in the applications envisaged.


Observations on the epicardial activation of the normal human heart. Serial hand mapping techniques in man have identified 3 to 5 sites of epicardial breaktrough (EBT). However, transmural epicardial excitation from the widely distributed His/Purkinje system suggests a more complicated pattern may exist. Multielectrode arrays used with large mapping systems during surgery often present complicated and sometimes inconsistent activation patterns. The purpose of this work is to reconcile epicardial activation in the normal human heart with anatomical and endocardial/intramural physiological recordings using multichannel computer mapping requiring only a single beat, and rigorously defined and applied activation time detection algorithms. Eighteen subjects undergoing surgery for Wolff-Parkinson-White syndrome were recorded with a 119 site sock array during nonpreexcited sinus rhythm. None had evidence of coronary artery disease and all exhibited a normal 12-lead ECG except during periods of preexcitation or tachycardia. Each was recorded bipolarly and four also were recorded monopolarly. Recordings revealed 8.0 +/- 1.6 EBTs (range 5 to 12). Closely spaced, multiple EBTs often were observed and usually confirmed using different activation time detection algorithms. The earliest EBT always occurred over the anterior right ventricle at 14.3 +/- 6.5 msec (range -1 to 29 msec) after QRS onset. Subsequent EBTs could occur at any ventricular site with variable latencies. In contrast to previous reports describing epicardial spread of activation from a few foci, a mosaic of epicardial activation emerges. These data are consistent with endocardially initiated transmural activation of the epicardium suggested by the anatomy of the His/Purkinje system and intramural recordings.


Atrial flutter--update on the mechanism and treatment. Atrial flutter is a common and usually benign but symptomatic supraventricular tachycardia. There is a striking similarity between patients with atrial flutter suggesting a common substrate despite the presence or absence of underlying heart disease. In man, the mechanism is a single reentrant circuit originating in the right atrium whose center appears to be functional within the anatomical constraints of the right atrium. The reentrant circuit of atrial flutter contains an area of slow conduction in the inferior right atrium but the size and exact location is uncertain. Drug therapy directed at terminating and preventing atrial flutter has been available for many years. The efficacy and safety of this therapy is not as well tested as is the same therapy for atrial fibrillation. The most effective way to terminate atrial flutter is a nonpharmacological approach. Several nonpharmacological methods provide new treatment options in the management of patients with drug resistant or hemodynamically unstable atrial flutter. The use of anticoagulation for this disorder is still evolving. There is a risk of clinically apparent thromboemboli in some patients with atrial flutter although the risk appears less than that for atrial fibrillation. In the future, refinements and improvements in therapy for atrial flutter will likely be derived from a better understanding of its mechanism.


Postpausal ventricular fibrillation analyzed by Holter-ECG: a case report. A 70-year-old male patient with a myocardial infarction, 3 months previously, happened to be wearing a Holter monitor at cardiac arrest during micturition. The patient had ventricular fibrillation (VF) and was successfully resuscitated by means of defibrillation. Analysis of the Holter tape revealed atrial fibrillation and secondary ventricular premature beats (VPB), i.e., VPBs dependent upon a relatively long interval between the two preceding heartbeats. The VF was preceded by an especially long R-R interval (1.45 sec) following a run of especially short R-R intervals implying the sharpest deceleration of the ventricular rate during 1 hour Holter recording preceding the cardiac arrest. A peculiar large alteration of the T wave configuration was also found to precede the VF.


NASPE Young Investigator Awardee 1992. Three-dimensional electrogram mapping improves ablation of left-sided accessory pathways. Conventional electrogram mapping techniques for localization of accessory pathways during radiofrequency ablation procedures are time consuming and often inaccurate. We hypothesized that a computer generated, three-dimensional electrogram of retrograde atrial activation created from signal-averaged sequential endocardial bipolar electrograms (collected from the atrial aspect of the mitral annulus using a single transseptal catheter and then time aligned to a known myocardial activation reference) would improve left-sided accessory pathway atrial insertion site identification and increase ablation efficiency. Ablation efficiency was defined by procedure time, fluoroscopy time, duration of radiofrequency energy required to achieve initial accessory pathway block, cumulative ablation energy per procedure, and number of radiofrequency energy applications. Patients with single left-sided accessory atrioventricular connections were studied. Standard mapping results in 31 patients (group A) were compared to a three-dimensional electrogram approach used in 26 patients (group B). Three-dimensional electrogram mapping reduced procedure time (group A 3.8 +/- 1.6 vs group B 2.8 +/- 0.9 hours, P < 0.004), fluoroscopy time (group A 45.3 +/- 35.0 vs group B 25.1 +/- 10.5 min, P < 0.02), time to accessory pathway block (group A 2.6 +/- 1.5 vs group B 1.2 +/- 0.5 sec, P < 0.002), cumulative radiofrequency energy (group A 2126 +/- 2207 vs group B 636 +/- 586 joules, P < 0.0008), and radiofrequency energy applications (group A 5.0 +/- 4.4 vs group B 1.7 +/- 1.2, P < 0.0002). We conclude that three-dimensional electrogram mapping improves left-sided accessory pathway atrial insertion localization, reduces ablation procedure time and radiation exposure, and improves ablation efficiency.


Effects of maternal smoking upon neuropsychological development in early childhood: importance of taking account of social and environmental factors. Data from a prospective study of 548 children followed from birth to 4 years of age were analysed to determine whether maternal smoking during and/or after pregnancy affects children's neuropsychological development. The differences in mean developmental test scores between children whose mothers smoked and those of mothers who did not smoke were slight, with subscale scores only 2.4 to 4.1% lower in children whose mothers smoked. These differences were not statistically significant after adjustment for socio-economic status, quality of home environment and mother's intelligence, suggesting that the social and environmental factors are major confounders of the association of exposure to maternal smoking and neuropsychological development in childhood. In order to gain a better understanding of this area, more precise measures of exposure to environmental tobacco smoke and comprehensive consideration of confounders will be required.


Normal development of nerve-muscle synapses in mice lacking the prion protein gene. The expression of acetylcholine receptors (AChR) at neuromuscular synapses in skeletal muscle is regulated by innervation. Recent evidence suggests that the neurotrophic factors involved in the expression of AChR subunit genes may be related to the prion protein (PrPc), a protein of unknown function expressed primarily in neurons which, in its modified form, PrPSc, is thought to have a role in the pathogenesis of transmissible spongiform encephalopathies. We have tested for an involvement of PrPc in the neurotrophic regulation of synaptic AChRs in muscle by comparing the contents of AChR epsilon- and gamma-subunit mRNAs by Northern blot analysis and by in situ hybridization in mice with normal and with deleted PrP genes. At the protein level, AChR expression was assessed electrophysiologically. No difference was found between muscles from the two types of animals, suggesting that the neural regulation of AChR subunit expression in skeletal muscle can be mediated by factors that are not derived from the PrP gene.


A patch-clamp study of GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons. GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons were studied by using patch-clamp techniques. Current-voltage relations for both agonists in symmetrical Cl- solutions showed outward rectification. Strychnine-sensitive glycine-activated currents have not been studied in detail before in post-natal tissue-cultured hippocampal neurons. Partial desensitization of whole-cell currents was observed in symmetrical Cl- solutions during bath application of GABA- and glycine. In F-/Cl- solutions, both agonists gave a PF/Pcl value of about 0.06. The reversal potentials in mixtures of Cl- and SO4(2-) solutions were close to the equilibrium potentials of Cl- in the presence of both GABA and glycine. Single channels in inside-out excised patches with 2 mumol l-1 GABA and 5 mumol l-1 glycine in the pipette showed outward current rectification. The dose-response curves for GABA and glycine gave Kd values of 52 and 61 mumol l-1, respectively, and Hill coefficients close to 2. GABA and glycine binding were competitively blocked by their respective antagonists (bicuculline and strychnine). The similarities between GABA- and glycine-activated currents and the response in a combination of saturating concentrations of both GABA and glycine implied that the two agonists activated comparable numbers of anatomically distinct channels with very similar permeation properties.


Structural studies on the photoreceptor phytochrome: reevaluation of the epitope for monoclonal antibody Z-3B1. The photoreceptor phytochrome is widely distributed in the plant kingdom from angiosperms to ferns, mosses and algae. The epitope for the monoclonal antibody Z-3B1 which exhibits wide-ranging cross-reactivity with phytochromes from higher and lower plants was mapped by the combination of several methods: by Western blot with proteolytic fragments of known localization, by sequence comparison of phytochromes from various plants, and by production of overlapping fusion proteins. The only sequence which is common to all positively-reacting fusion proteins is the sequence A-830 to R-859. This sequence must contain the Z-3B1 epitope. The best candidate is suggested to be the T-cell antigenic sequence K-Y-V/I-E-A/C-L-L-T (= K-848 to T-855). The significance of the highly conserved epitope in all phytochromes is discussed.


Production of monoclonal antibodies to human immunodeficiency virus type-2. The ROD strain of the human immunodeficiency virus type 2 (HIV-2) was used to produce monoclonal antibodies. Virus grown in CEM cells was partially purified by ultracentrifugation and solubilized in a buffer containing Triton X-100. BALB/c mice were inoculated intraperitoneally with 50 micrograms of solubilized virus preparations mixed 1:1 with complete Freund's adjuvant. Animals were boosted on day 28 and sacrificed on day 31. Spleen cells from the immunized animals were fused with SP20/Ag 14 myeloma cells and cultured in HAT medium. Following selection of the hybrids of interest by an HIV-2 ELISA procedure, hybridomas were cloned twice by limiting dilution. Six clones were found to produce antibodies that reacted with HIV-2 antigens as judged by ELISA. These antibodies were concentrated by ammonium sulfate precipitation, and analyzed by the Western blot procedure. Monoclonal antibodies specifically reactive to an HIV protein of 68 KD were obtained. These antibodies did not react with an HIV-2 band of 55 KD. These data showed that the monoclonal antibodies recognized the carboxy terminal region (the RNAse H domain) of the HIV-2 retrotranscriptase enzyme.


[Interferon and its potential use in the treatment of infectious diseases]. Modern recombinant biotechnology has made possible the production of large amount of interferons and their use as immunotherapeutic agents. Most of the biological, physical and chemical characteristics of interferons has been established, including their classification, genetic structure, chemical composition and possible mechanisms of action. Interferons have been utilized in clinical studies with human and experimental animals against bacterial, mycotic, parasitic and viral infections. Success has been reported mainly when administered prophylactically against acute infections. Favorable results have been obtained, both prophylactic and therapeutically, in some chronic diseases and in those in which the microorganism has an intracellular phase during its life cycle. Moreover, a promising future has been suggested for the combined use of interferon with other antimicrobial drugs.