Enhanced hprt mutant frequency but no significant difference in mutation spectrum between a smoking and a non-smoking human population. Recently, we have observed a small (36%), but significant, enhancement of the frequency of 6-thioguanine (6-TG)-resistant T-lymphocytes in blood from smokers. The molecular nature of 43 hypoxanthine-guanine phosphoribosyltransferase (hprt) mutant T-lymphocyte clones from nine smoking individuals was determined to investigate whether the increase in hprt mutant frequency would lead to a changed mutation spectrum. The types and distribution of hprt mutations in smokers was compared with those found in 55 6-TGr T-lymphocyte clones from 12 members of a control group of non-smokers. From this control group 25 hprt mutants were novel, whereas 31 have been described previously. Among smokers and non-smokers, a similar proportion of base substitutions (approximately 35%), mutations causing aberrant splicing (approximately 37%), frameshifts (approximately 16%) and deletions (approximately 9%) was found. In both groups, GC----AT base pair changes were found to be predominant among transitions. However, whereas all types of transversions were about equally represented in non-smokers, GC----TA transversions were not recovered among smokers. Investigation of the distribution of base substitutions over the hprt coding region showed no differences between the two groups. These data provide no clues on the nature of DNA adducts induced by smoking, which are thought to be responsible for the increased mutation frequency at the hprt locus in T-lymphocytes from smokers.


Species differences in urinary butadiene metabolites; identification of 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, a novel metabolite of butadiene. 1,3-Butadiene (BD) is used in the manufacture of styrene-BD and polybutadiene rubber. Differences seen in chronic toxicity studies in the susceptibility of B6C3F1 mice and Sprague-Dawley rats to BD raise the question of how to use the rodent toxicology data to predict the health risk of BD in humans. The purpose of this study was to determine if there are species differences in the metabolism of BD to urinary metabolites that might help to explain the differences in the toxicity of BD. The major urinary metabolites of BD in F344/N rats, Sprague-Dawley rats, B6C3F1 mice, Syrian hamsters, and cynomolgus monkeys were identified as 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (I) and the N-acetylcysteine conjugate of BD monoxide [1-hydroxy-2-(N-acetylcysteinyl)-3-butene] (II). These mercapturic acids are formed by addition of glutathione at either the double bond (I) or the epoxide (II) respectively. When exposed to approximately 8000 p.p.m. of BD for 2 h, the mice excreted 3-4 times as much metabolite II as I, the hamster and the rats produced approximately 1.5 times as much metabolite II as I, while the monkeys produced primarily metabolite I. The ratio of formation of metabolite I to the total formation of the two mercapturic acids correlated well with the known hepatic epoxide hydrolase activity in the different species. These data suggest that (i) the availability of the monoepoxide for conjugation with glutathione is highest in the mouse, followed by the hamster and the rat, and is lowest in the monkey; and (ii) the epoxide availability is inversely related to the hepatic activity of epoxide hydrolase, the enzyme that removes the epoxide by hydrolysis. The ratio of the two mercapturic acids in human urine following BD exposure may indicate the pathways of BD metabolism in humans and may aid in the determination of the most appropriate animal model for BD toxicity.


Dietary hydrogen peroxide enhances hepatocarcinogenesis in trout: correlation with 8-hydroxy-2'-deoxyguanosine levels in liver DNA. The tumor-enhancing effect of hydrogen peroxide (H2O2) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated rainbow trout hepatocarcinogenesis was investigated and correlated with the levels of the mutagenic DNA adduct 8-hydroxy-2'-deoxyguanosine (oh8dG). In addition, the protective role of vitamin E was examined in relation to tumor enhancement and oh8dG levels in liver DNA. Trout were fed diets containing two levels of vitamin E (1000 or 20 mg/kg wet wt), each of which were made up to contain three levels of H2O2 (0, 600 or 3000 p.p.m.). Dietary vitamin E levels had no significant effect on tumor incidence or levels of oh8dG in liver DNA. On the other hand, dietary H2O2 enhanced liver tumors in a dose-dependent manner. Liver tumor incidence correlated significantly with the mean level of liver DNA oh8dG content (r = 0.87). We conclude that the H2O2 tumor-enhancing effect coincides with higher levels of oh8dG in the trout liver genome. Thus, rainbow trout may be a useful model for the study of the relationship of oh8dG levels in vivo to enhancement or promotion of carcinogenesis and its modulation by dietary enhancers and inhibitors of oxidative stress.


Inhibition of repairable DNA-damage in Escherichia coli K-12 cells recovered from various organs of nitrosamine-treated mice by vitamin A, phenethylisothiocyanate, oleic acid and triolein. The influence of various dietary constituents--phenethylisothiocyanate (PEITC), oleic acid (OA), triolein (TO), and vitamin A (ROL)--on the genotoxic activity of nitrosamines (NDMA, NDELA, NPYR) was investigated. For this purpose differential DNA repair assays with Escherichia coli K-12 strains were performed in vitro and in vivo with mice. Under in vitro conditions (liquid holding), all compounds reduced nitrosamine induced DNA-damage in the indicator bacteria in the dose range 1-10 micrograms/ml, the ranking order of efficiency being PEITC greater than OA greater than ROL greater than or equal to TO. In animal-mediated assays, acute oral treatment with PEITC (17-150 mg/kg), 2 h before nitrosamine administration, resulted in a marked decrease of nitrosamine genotoxicity in liver, kidneys, lungs and in the blood. Also in other organs (spleen, testes) an increase in differential survival (which serves as a measure for repairable DNA damage) occurred. With ROL only a comparatively moderate antigenotoxic effect was obtained at a high dose level (250 mg/kg) under identical experimental conditions. OA (2000 mg/kg) and TO (16,000 mg/kg) were completely inactive. Upon repeated treatment (consecutive oral administration of the putative antigenotoxins over 4 days, a final treatment 24 h before nitrosamine administration) PEITC (150 mg/kg/day), ROL (80 mg/kg/day) and OA (2000 mg/kg/day) had no influence on the genotoxic effects of the nitrosamines. Repeated treatment with TO (4000-16,000 mg/kg/day) resulted in a moderate dose-dependent reduction of NDMA-induced DNA-damage in the indicator bacteria, whereas in combination with NPYR only a marginal effect was observed. Biochemical experiments indicated that the antigenotoxic effects of PEITC seen under in vivo conditions were due to inhibition of alpha-hydroxylation of the nitrosamines, whereas ROL and TO appeared not to interfere strongly with this metabolic activation step. Our results indicate that in vitro assays do only partly reflect the antigenotoxic properties of the different food constituents in vivo and that animal-mediated DNA repair assays with E. coli strains are an appropriate approach to study the effects of modifiers of nitrosamine genotoxicity in the living animal.


Ras involvement in cells transformed with 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) in vitro and with N-[4-(5-nitro-2-furyl)-2-thiazoyl]formamide in vivo. N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) administration to rats followed by sodium saccharin results in transitional cell carcinomas of the bladder, of which 24% harbor an activated H-ras gene. Since 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) is the mutagenic and carcinogenic metabolite of FANFT in vivo, we wished to examine ras activation in in vitro ANFT-transformed rat bladder epithelial cells as well as four cell lines established in culture from in vivo FANFT-induced rat bladder tumors. Screening by Western blotting revealed no enhanced levels of p21ras in ANFT-transformed cells nor in cells established in culture from FANFT-induced rat bladder carcinomas. Further investigations using immunohistochemical staining with a different pan-reactive p21 monoclonal antibody (Cetus Corporation) specific for this method, however, showed two groups of cells from FANFT-induced rat bladder tumors had enhanced immunoreactivity. Apart from this, p21ras expression of most of the cells groups varied little from the controls. We examined the reported hot spots (exons 1 and 2) of each of the ras genes (H-, K- and N-ras) by direct sequencing of amplified DNA. No mutations were present. We conclude, therefore, that ANFT transformation of primary rat bladder epithelial cells in vitro may not in this case be mediated by ras activation, although this is difficult to determine since others have observed that optimal culture conditions can select for certain populations of cells without ras activation.


Formation and 32P-postlabeling of DNA and tRNA adducts derived from peroxidative activation of carcinogenic azo dye N,N-dimethyl-4-aminoazobenzene. Peroxidase in the presence of hydrogen peroxide catalyzes in vitro the activation of carcinogenic N,N-dimethyl-4-aminoazobenzene (DAB) to DNA-, tRNA- and homopolydeoxyribonucleotide-bound products. tRNA is the most susceptible to modification by the activated DAB. Binding of DAB products to macromolecules is inhibited by methyl viologen, nitrosobenzene, ascorbate, glutathione, NADH and MgCl2. The mechanism of these inhibitions was studied. The nuclease P1 version of the 32P-postlabeling assay was employed for detection and quantitation of some major DNA or tRNA adducts formed with DAB activated by a peroxidase system. tRNA modified by activated DAB shows a significantly increased acceptance for L-methionine.


Dose-response study on covalent binding to forestomach protein from male F344 rats following oral administration of [14C]3-BHA. A dose-response study on covalent binding to forestomach protein was performed using male F344 rats following oral administration of [14C]3-tert-butyl-4-hydroxyanisole (3-BHA). The order of tissue distribution of radioactivity 6 h after oral administration of 1% [14C]3-BHA was forestomach greater than glandular stomach greater than liver greater than kidney greater than plasma. The covalent binding levels to forestomach protein were very low until 0.1% 3-BHA, but rapidly increased at concentrations of 1% and 2% 3-BHA. The dose-response relations of 3-BHA levels to the covalent binding to protein coincided well with the incidence of forestomach papilloma reported previously. The binding levels of forestomach and glandular stomach were compared. In case of i.v. administration, both binding levels were almost the same, however, in case of 0.1% p.o. administration, the forestomach level was approximately 8-fold higher than the glandular stomach level. The binding level of forestomach protein by p.o. administration was approximately 54-fold higher than that by i.v. administration. Although the amount of tert-butylhydroquinone (BHQ) was very low compared with the amount of covalent binding, the BHQ levels in forestomach were dependent upon the dose levels of 3-BHA. Our study indicates that the dose-response study on covalent binding to target tissue protein is an efficient method for the quantitative estimation of the active metabolites coming from the chemicals which form the quinone metabolites.


Altered cellular calcium regulation and hepatic glucose production during hemorrhagic shock. The relationship between intracellular Ca2+ and glucose production in the liver during early and late states of hemorrhagic shock was studied. Rats were anesthetized with intraperitoneal sodium pentobarbital and both femoral arteries and one femoral vein were cannulated. Rats were divided into two groups. One group was subjected to hemorrhagic shock by rapid withdrawal of blood to a mean arterial pressure of 40 mm Hg and maintained in shock for either 30 or 150 min. Rats in the control group were observed for the same time period. Hepatic glucose production was evaluated in both groups by a nonrecirculating liver perfusion model with and without lactate as a substrate. Intracellular free Ca2+ in hepatocytes was measured using the Ca2+ selective indicator Fura-2, under basal and epinephrine-stimulated conditions. Hyperglycemia and hyperlacticacidemia were observed in vivo at 30 min of hemorrhagic shock, whereas hypoglycemia and hyperlacticacidemia were observed at 150 min of shock. Hepatic glucose production in isolated perfused livers was significantly depressed at 30 min in animals subjected to shock (P less than 0.05). Lactate-induced glucose production was significantly attenuated at 30 and 150 min (P less than 0.05). Basal Ca2+, in isolated hepatocytes, at 30 and 150 min of hemorrhagic shock was significantly (P less than 0.05) higher than in controls. The hemorrhagic shock rat hepatocytes failed to evaluate intracellular free Ca2+ upon stimulation with 10(-5) M epinephrine. These results demonstrate that hemorrhagic shock is associated with an increase in hepatocyte intracellular Ca2+ concentration along with attenuation of hormone-mediated mobilization of calcium and substrate specific stimulation of hepatic glucose production.


Expressed emotion in parents of non-referred children aged 6 to 11 years from two school populations: a pilot study. This study investigates the role of parental expressed emotion (EE) in families of well-adjusted children. These were randomly selected from two school populations representing two different socio-economic backgrounds. Maternal criticism was strongly correlated with children's behaviour, although both the EE ratings and the scores of children's behaviour were within the established normal range. Mothers were more emotionally over-involved but also more positive towards their children in manual families. Fathers expressed more warmth towards boys. Clinical and methodological issues regarding the concept of EE are discussed.


Early gut ischemia in experimental fecal peritonitis. Tissue oxygenation in the gastrointestinal tract was studied in a porcine model in which septic shock was induced by fecal peritonitis. The oxygen delivered was estimated by measuring the portal venous blood flow and the calculated arterial oxygen saturation. The oxygen consumption of the gut, including the pancreas and spleen, was monitored by measuring the portal venous blood flow and the difference between the calculated arterial oxygen and the measured portal venous oxygen saturation. In addition, the oxygenation of the gut mucosa was followed via the tonometric technique. Furthermore, lactate was measured in arterial and portal blood. The experimental animals were divided into two groups, one control (n = 6) and one experimental (n = 6). Peritonitis was introduced by installation of a standardized amount of autologous feces into the abdominal cavity. The animals were followed for 5 hr. Very early during the course of sepsis there was a fall in gut intramucosal pH (pHi), and this was evident before any reduction in splanchnic DO2. Furthermore, an early increase in splanchnic VO2 was evident simultaneously with the fall in pHi. Arterial pH and lactate were not able to detect the inadequate regional tissue oxygenation. It is concluded that pHi measured with the tonometric technique is sensitive in detecting gut mucosal ischemia, and it is therefore highly likely that tonometry would be a valuable method in monitoring severe ill patients.


Service needs of families of children with severe physical disability. Service contacts, perceived helpfulness of services and needs for help were investigated in a sample of 107 families of young children with severe physical disability. Many families were in contact with a multiplicity of different services and overall frequency of contact was high. Despite this, there was evidence of considerable unmet need, particularly in the provision of information to families. Families with the highest levels of unmet need were likely to have experienced high levels of strain from life events and to have children with mental retardation as well as physical disability, fathers in those families were more likely to be unemployed and mothers were more likely to use passive optimism in coping with child problems. The findings indicate the importance of services which are easily accessible to parents, the provision of information to parents about such services, the co-ordination of services through a 'link' person and the accurate and individual assessment of family needs.


Effect of platelet-activating factor antagonist and leukotriene antagonist on endotoxin shock in the rat: role of the leukocyte. The effects of platelet-activating factor (PAF) antagonist (CV-3988) and leukotrienes (LTs) antagonist (ONO-1078) on endotoxin-induced sequelae in the rat were assessed. Pretreatment with either CV-3988 (6 mg/kg, i.v.) or ONO-1078 (150 mg/kg, p.o.) did not improve survival rate following the administration of Escherichia coli lipopolysaccharide (LPS) compared with that of control rats pretreated with solvents of the drugs. Rats pretreated with both CV-3988 and ONO-1078 exhibited significantly (P less than 0.01) enhanced survival following lipopolysaccharide (LPS) administration. Percentage survivals 48 hr after the administration of LPS were 20%, 32%, 24%, and 68% in the pretreatment with solvents, CV-3988, ONO-1078, and CV-3988 combined with ONO-1078 groups, respectively. Pretreatment with CV-3988 combined with ONO-1078 inhibited the change of plasma transaminase 3 hr after LPS administration. The neutropenia, due to the administration of vinblastine (1 mg/kg, i.v.), increased the survival rate following the administration of LPS without pretreatment with PAF and LT antagonists. Antishock action of CV-3988 and ONO-1078 could not be seen in neutropenic rats. These data suggest that combined pretreatment with PAF antagonist and LT antagonist inhibited leukocyte-mediated tissue injury in LPS-induced endotoxemia.


Impaired motor skill (clumsiness) in otherwise normal children: a review. Some children, who are otherwise normal, experience unusual difficulties with the acquisition and performance of motor skills. These children are commonly described as being clumsy. Impaired performance of motor skills, to the degree experienced by clumsy children, is unlikely to present a serious problem. However, a review of the literature suggests that significant associated and secondary emotional problems are common. In particular, such problems are likely to result in children not achieving their full potential. Therefore, it is important that the cause of affected children's impaired motor skills be recognized early so that these problems can be avoided, or at least minimized, by a sympathetic understanding of their difficulties. The problem is that, whilst severely affected children can be readily recognized, identification of mildly and moderately clumsy children is difficult.


Parental involvement in the KIDS Family Centre: who does it work for? Although parental involvement has been a major development in the field of disability and handicap, there has been relatively little systematic investigation of the value and appreciation of parental involvement from the parent's point of view. This study addresses the use and appraisal of services by parents at the KIDS Family Centre, Camden, London, which offers a variety of family-focused services with differing degrees of parental involvement. The evidence showed that the overall appraisal of the Centre was high but, given the choice, individual parents selected and appreciated different kinds of parental involvement services. Certain family background variables were associated with the pattern of selection and rating.


Mechanism of immunoreactive atrial natriuretic factor release in an ovine model of endotoxemia. We have previously reported an increase in plasma levels of atrial natriuretic factor (ANF) in an ovine model of endotoxemia. The purpose of this study was to determine if this IR-ANF release was mediated by the increase of right atrial pressure (RAP) and right heart volumes concomitantly observed following endotoxin (LPS) administration. We studied right ventricular function, renal blood flow (RBF), urinary output (UO), urinary clearance of free water (CH20), urinary osmolality (UOSM), sodium excretion (UENA), and the plasma IR-ANF concentration (radioimmunoassay), following the administration of an E. coli LPS bolus (1 microgram/kg) with (group O, n = 8) and without (group E, n = 10) pretreatment with OKY-046, a selective thromboxane synthetase inhibitor. LPS induced early increases in RAP, right ventricular end-systolic (RVESV) and end-diastolic (RVEDV) volumes, heart rate (HR), and IR-ANF, and delayed increases in RBF, UO, and CH20. OKY-046 prevented the elevation of RAP, RVEDV, and RVESV; however, both groups showed virtually identical increases in IR-ANF (E: 20.03 +/- 3.8 to 192.33 +/- 35.47 pg/ml, O: 17.9 +/- 4.1 to 159.5 +/- 23 pg/ml) as well as an increase of HR, RBF, UO, and CH20. The increase in IR-ANF release noted following the administration of LPS in an ovine model does not appear to be related to the early elevations in right heart volumes or atrial distension.


Effect of sepsis on intracellular sodium activity, sodium concentration, and water content in thermal injured rat. The effects of sepsis on intracellular Na+ activity, Na+ concentration, and H2O partition in skeletal muscle were investigated in a burn rat model. Studies were performed on either postburn day 3 or day 7 during evolving burn wound sepsis. Data are compared among 3 groups of rats: burned and infected (BI), burned not infected (B), and sham burn (C). After 3 days postburn both Na+ activity and concentration decreased in the BI group as compared with B and C groups. By postburn day 7, the BI group developed septic shock and had increased intracellular Na+ activity and concentration. The resting membrane potentials of skeletal muscle cells depolarized. The finding of an increased cell membrane relative permeability of Na+ to K+ could account for the increase in Na+ influx into cells. In addition, intracellular and total muscle H2O contents decreased and extracellular H2O increased. Hypernatremia, hyperchloremia, and hyperosmolality were also observed in the BI group. However, the fact that there was no significant difference between B and C groups indicates that the late derangements were due to septic shock rather than simple burn injury. Thus, the deleterious effects of the evolving burn wound sepsis on Na+ homeostasis might be due to the detrimental effect of increased intracellular Na+ activity on mitochondrial respiratory control with subsequent impairment of cellular functions.


Review: neurotoxicity of lead.

Review: neurotoxicity of lead. 'Lead and its compounds are potentially toxic; the element has no known physiological function; it is widely distributed in nature and as a result of man's activities' (Lawther Report: DHSS 1980). Concern grew in the late 1970s that even low levels of exposure to lead caused adverse changes in children's adjustment and academic attainment. This paper traces the rapid developments of the past decade in investigating the relationship between body lead burden and relevant outcome measures. Research strategies moved from small-scale, clinical-descriptive studies to large-scale epidemiological studies; from cross-sectional studies to longitudinal ones. Agreement had to be reached on how to measure body lead burden. Decisions had to be made on which outcome measures to use. Measures of global intelligence were complemented by measures of academic attainment, emotional adjustment and hyperactivity, as well as by experimental measures of more basic psychophysiological functioning. Ways had to be found of dealing experimentally and statistically with socioeconomic factors which acted as confounds. The investigations were marked by tremendous collaboration between investigators, between national agencies and across countries. The methodological issues are relevant to the investigation of other toxins and they define a field of developmental behavioural toxicology. This case study can act as a model for investigating the effects of other environmental toxins and hazards.


Effect of endotoxicosis on plasma and tissue levels of calcitonin gene-related peptide. This study was designed to investigate the changes in tissue content and plasma concentrations of CGRP, a 37 amino acid vasoactive peptide, in male Sprague Dawley rats injected intravenously with a nonlethal dose of 3 mg/kg E. coli endotoxin. Plasma CGRP concentrations in nonendotoxemic animals, measured by a specific RIA, were initially 30.5 +/- 3.3 pg/ml, and were significantly increased to 63.7 +/- 4.6 pg/ml 2 hr after induction of endotoxemia (P less than 0.001; n = 13). A higher dose of LPS did not further elevate plasma CGRP levels, indicating that the maximal response occurred following a dose of 3 mg/kg LPS. CGRP levels in abdominal aorta, inferior vena cava, stomach, kidney, and left ventricular myocardium (4.11, 8.5, 2.61, 0.69, and 0.25 pmol/g wet weight tissue, respectively) were not changed significantly following the injection of endotoxin. However, in lung and mesenteric artery the levels increased significantly from 1.47 +/- 0.12 and 7.97 +/- 1.32 pmol/g wet weight tissue to 1.96 +/- 0.19 (P less than 0.05, n = 11) and 15.02 +/- 2.3 pmol/g (P less than 0.01; n = 7), respectively. In contrast, CGRP levels in the duodenum were significantly decreased from 11.3 +/- 0.93 pmol/g wet weight tissue to 6.2 +/- 0.68 pmol/g (P less than 0.001; n = 6). The changes in plasma concentration and tissue content of CGRP suggest that splanchnic organs may be the source of the elevated plasma CGRP levels in endotoxemia and that selective organ CGRP levels reflect a role in the pathogenesis of the response to endotoxemia.


Reactivity of monoclonal antibody E5 with endotoxin. I. Binding to lipid A and rough lipopolysaccharides. The murine IgM monoclonal antibody (mAb) E5 was produced by a hybridoma derived from spleen cells of a mouse immunized with the J5 rough mutant of Escherichia coli O111:B4. In a multicenter randomized placebo-controlled clinical trial, E5 has been shown to reduce significantly the mortality and morbidity of patients with Gram-negative sepsis. The characteristics of E5 binding to endotoxin were studied in vitro. We report here the results of binding to an extensive panel of rough lipopolysaccharide (LPS) and lipid A preparations. Using standard immunologic techniques, including enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), as well as an antibody capture assay using immobilized antibody and a chromogenic Limulus amebocyte lysate (LAL) detection system, E5 was shown to bind to all rough LPS (chemotypes Ra through Re from Salmonella minnesota and E. coli J5) and lipid A preparations tested. E5 displayed a Kd for Ra LPS of approximately 6.5 nM. These results confirm and extend those reported previously and provide evidence that E5 binds specifically to lipid A and to the lipid A moiety of rough LPS.


In vivo assessment of regional microvascular albumin leakage during E. coli septic shock in the baboon model. Changes in regional microvascular albumin flux during septic shock were studied noninvasively by scintigraphy in the baboon model. Use was made of an i.v. injection of 99mTc-labeled baboon serum albumin. Count ratios of lung to cardiac, liver to cardiac, and abdominal to cardiac regions were measured two-hourly for 6 hr in six control and six septic shock baboons (live E. coli) and compared. Increased ratios obtained during shock pointed to an increase in extravascular albumin. Linear regression lines fitted to these count ratios provided regional albumin leak indices. These indices demonstrated statistically significant increases (P less than 0.05) during septic shock for the abdominal region during the 6-hr study, and for all regions, but especially the abdomen, when data were calculated over 4 hr. Increasing ratios and leak indices correlated with postmortem data and with changes in neutrophil and platelet behaviour previously established during shock. Possible accompanying mediator releases could be responsible for the endothelial damage leading to the increased permeability.


Mouse phospholamban gene expression during development in vivo and in vitro. To establish a murine model that may allow for definition of the precise role of phospholamban in myocardial contractility through selective perturbations in the phospholamban gene, we initiated studies on the role of phospholamban in the murine heart. Intact beating hearts were perfused in the absence or presence of isoproterenol, and quantitative measurements of cardiac performance were obtained. Isoproterenol stimulation was associated with increases in the affinity of the sarcoplasmic reticulum Ca2+ pump for Ca2+ that were due to phospholamban phosphorylation. To assess the regulation of phospholamban gene expression during murine development, Northern blot and polymerase chain reaction analyses were used. Phospholamban mRNA was first detected in murine embryos on the ninth day of development (the time when the cardiac tube begins to contract). In murine embryoid bodies, which have been shown to recapitulate several aspects of cardiogenesis, phospholamban mRNA was detected on the seventh day (the time when spontaneous contractions are first observed). Only those embryoid bodies that exhibited contractions expressed phospholamban transcripts, and these were accompanied by expression of the protein, as revealed by immunofluorescence microscopy. Sequence analysis of the cDNA encoding phospholamban in embryoid bodies indicated complete homology to that in adult hearts. The deduced amino acid sequence of murine phospholamban was identical to rabbit cardiac phospholamban but different from dog cardiac and human cardiac phospholamban by one amino acid. These data suggest that phospholamban, the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, is present very early in murine cardiogenesis in utero and in vitro, and this may constitute an important determinant for proper development of myocardial contractility.


Cellular mechanisms for synthesis and secretion of atrial natriuretic peptide and brain natriuretic peptide in cultured rat atrial cells. To investigate the cellular mechanism for the synthesis and secretion of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), we examined the effects of vasoactive agents on the secretion rates and gene expression of ANP and BNP in cultured rat atrial cells. Endothelin (10(-7) M, +61%), 12-O-tetradecanoylphorbol 13-acetate (TPA, 10(-6) M, +62%), the calcium ionophore A23187 (10(-6) M, +95%), and Bay K 8644 (10(-6) M, +34%) (p < 0.05 each) all increased the secretion of ANP into the culture media in a dose-dependent fashion. On the other hand, endothelin (10(-7) M, +57%) and TPA (10(-6) M, +55%) (p < 0.01 each) increased the secretion of BNP in a dose-dependent manner, whereas A23187 (10(-6) M, -45%, p < 0.001) suppressed the secretion of BNP in a dose-dependent manner, and Bay K 8644 caused no significant effects on BNP secretion. The molecular forms of intracellular ANP were exclusively gamma-ANP, whereas those of BNP were gamma-BNP and its carboxy terminal 45-amino-acid peptide, BNP-45. The ratio of media to cell contents was much higher in BNP than in ANP. Northern blot analysis revealed that both ANP mRNA and BNP mRNA levels were significantly increased by 10(-7) M endothelin (ANP mRNA, +52%; BNP mRNA, +36%; p < 0.05 each) and 5 x 10(-5) M 1-oleoyl-2-acetylglycerol (ANP mRNA, +296%; BNP mRNA, +133%; p < 0.01 each) but not by 10(-6) M A23187. Thus, the secretion of ANP is stimulated by both the elevation of [Ca2+]i and the activation of protein kinase C, whereas its synthesis is increased mainly by the activation of protein kinase C. The synthesis and secretion of BNP are augmented by the activation of protein kinase C rather than the elevation of [Ca2+]i. Furthermore, the processing and secretion of ANP and BNP may be regulated in different manners.


Brain ouabain-like activity and the sympathoexcitatory and pressor effects of central sodium in rats. Intracerebroventricularly infused hypertonic saline elicits sympathoexcitatory and pressor effects. To clarify the mechanisms mediating these effects, we evaluated blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA) responses to intracerebroventricular administration of 0.3 M NaCl, ouabain, and rat hypothalamic and pituitary extracts containing ouabain-like activity (OLA) in conscious Wistar rats, before and after intracerebroventricular preinjection of digoxin-specific antibody Fab (DAF) fragments. To exclude modulatory effects of arginine vasopressin (AVP), treatment with DAF fragments was in all experiments preceded by intravenous injection of the AVP antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,o- Me-Tyr2,Arg8]AVP. After AVP antagonist pretreatment, 0.3 M NaCl i.c.v. at 3.8 microliters/min for 10 minutes caused simultaneous increases in BP, RSNA, and HR. After AVP antagonist pretreatment, intracerebroventricular injections of 0.3 and 1.0 microgram/l microliter ouabain or the OLA equivalent to 1 microgram ouabain/2 microliters elicited similar significant increases in BP, HR, and RSNA. After pretreatment with AVP antagonist and DAF fragments (66 micrograms/4 microliters i.c.v.), BP, HR, and RSNA responses to 0.3 M NaCl, ouabain, and OLA were all significantly diminished. In contrast, combined AVP blockade and DAF fragments did not affect the BP response to intracerebroventricular angiotensin II, the BP, HR, and RSNA response to intracerebroventricular carbachol and to air stress, or the HR and RSNA responses to intravenous sodium nitroprusside. Intracerebroventricularly injected gamma-globulins (66 micrograms/4 microliters) did not affect the responses to 0.3 M NaCl, ouabain, or OLA.(ABSTRACT TRUNCATED AT 250 WORDS)


Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle. We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.


Sympathetic stimulation and norepinephrine infusion modulate extracellular potassium concentration during acute myocardial ischemia. The purpose of this study was to investigate whether sympathetic stimulation modulated the rise in extracellular K+ concentration ([K+]o) evoked by acute myocardial ischemia. In 35 alpha-chloralose-anesthetized dogs, we measured changes in [K+]o during acute myocardial ischemia in the presence and absence of sympathetic stimulation or norepinephrine infusion. A series of four 5-minute occlusions of the distal left anterior descending coronary artery (LAD) was completed in 18 dogs. Thirty minutes of reperfusion separated each LAD occlusion. Four to five K(+)-sensitive electrodes were inserted into the left ventricular midmyocardium that was perfused by the distal LAD. Lead II of the electrocardiogram, arterial pressure, and [K+]o were recorded, and the right atrium was paced at a constant cycle length. The first, second, and fourth LAD occlusions were done in the absence of sympathetic stimulation or norepinephrine infusion. The changes in [K+]o evoked by the first LAD occlusion differed (p < 0.05) from those elicited by the second and fourth occlusions. However, the changes in [K+]o during the second and fourth LAD occlusions were similar (p > 0.2) and served as controls for the responses obtained during the third occlusion. Two minutes before the third LAD occlusion, sympathetic stimulation (4 Hz) or norepinephrine infusion (0.25-0.5 micrograms/kg per minute i.v.) was begun and was continued until 2 minutes after reperfusion. We found that sympathetic stimulation and norepinephrine infusion increased (p < 0.05) myocardial blood flow in both normal and ischemic tissue. The mean response recorded by 23 K(+)-sensitive electrodes in 11 dogs showed that sympathetic stimulation increased (p < 0.001) the [K+]o at 1, 2, 3, 4, and 5 minutes after the onset of LAD occlusion compared with the second and fourth occlusions. In contrast, the mean response recorded by 20 K(+)-sensitive electrodes in seven dogs showed that norepinephrine infusion reduced (p < 0.02) the [K+]o at 4 and 5 minutes after the onset of LAD occlusion. These data show that sympathetic stimulation increased the [K+]o evoked by acute myocardial ischemia, an effect that was not mimicked by the intravenous administration of norepinephrine.


Adenosine A1 receptor activation attenuates cardiac injury produced by hydrogen peroxide. Adenosine has been shown to protect the ischemic and reperfused myocardium. To examine whether the protective effect of the nucleoside is mediated by modulation of oxidative stress, isolated rat hearts were perfused for 30 minutes with 100 microM H2O2 or an exogenous free radical-generating system consisting of purine (3.06 mM) and xanthine oxidase (10 units/l) in the presence or absence of drugs acting on adenosine A1 or A2 receptors. H2O2 alone produced a greater than 90% loss in contractility concomitant with a threefold elevation in resting tension, although these effects occurred in the absence of ultrastructural damage. Two A1 receptor agonists N6-cyclopentyladenosine (CPA, 1 microM) and R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA, 1 microM) significantly attenuated the cardiodepressant effects of H2O2 and depressed the elevation in resting tension; however, only the effect of CPA was found to be significant with regard to the latter parameter. A similar concentration of S(+)-N6-(2-phenylisopropyl)adenosine (S-PIA), a markedly less potent A1 receptor agonist, was found to be without beneficial effect. However, a significant protective effect against both the reduction in contractility and the elevation in resting tension was seen with a 10-fold elevation in the concentration of S-PIA (10 microM). The protective effects on functional parameters were associated with preservation of high-energy phosphate and adenine nucleotide contents after 30 minutes of H2O2 treatment. The salutary effects of all drugs were reversed in the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.5 microM). An A2 receptor agonist 2-[p-(carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine, termed CGS 21680 (1 microM), failed to alter the cardiac response to H2O2 with regard to all parameters studied. Neither a 50% reduction in external CaCl2 concentration nor treatment with 10 microM DL-propranolol exerted salutary effects against H2O2-induced dysfunction. None of the A1 receptor agonists modulated the response to purine plus xanthine oxidase. Our results demonstrate a selective protective effect of adenosine A1 receptor activation against the cardiac toxicity of H2O2 and provide, at least in part, a basis for the cardioprotective actions of adenosine and its analogues.


Myocardial energetics during ventricular fibrillation investigated by magnetization transfer nuclear magnetic resonance spectroscopy. Ventricular fibrillation (VF) is known to produce alterations in myocardial energetics, but the mechanism of these changes remains unclear. To investigate energy metabolism during VF, phosphorus nuclear magnetic resonance spectroscopy and magnetization transfer were applied to isolated perfused ferret hearts. VF was induced either by perfusion with digitalis (strophanthidin, 30 microM) or by high-frequency electrical stimulation. We measured the flux in two critical reactions: from inorganic phosphate (Pi) to ATP (ATP synthesis rate) and from phosphocreatine (PCr) to ATP (energy transfer capacity). During digitalis-induced VF, energy-related phosphates showed changes similar to those during hypoxia: myocardial [Pi] increased and [PCr] decreased. Concomitantly, the ATP synthesis rate increased to levels about threefold higher than control, whereas oxygen consumption increased by only 16%. The ATP synthesis rate exhibited a strong negative correlation with left ventricular pressure during VF (r = -0.95, n = 5, p < 0.02), whereas oxygen consumption did not (r = 0.19, p > 0.05). On the other hand, energy transfer capacity catalyzed by creatine kinase was significantly smaller during VF than in the control condition but still higher than the simultaneous ATP synthesis rate. In contrast to the marked energetic deterioration during VF induced by digitalis, electrically induced VF led to only a small increase in [Pi] and a small decrease in [PCr], and there were no significant changes in the ATP synthesis rate, energy transfer capacity, or O2 consumption. These results indicate that the rundown in energy metabolism during VF induced by digitalis was mainly attributable to a limitation of energy production through oxidative phosphorylation as well as to a marked increase in energy consumption. In contrast, myocardial energy generation remained unimpaired during VF induced by electrical stimulation. Intracellular calcium overload is more severe during VF induced by digitalis than during electrically induced VF (Circ Res 1991;68:1378-1389); severe calcium overload would be expected to compromise the capacity for energy generation by mitochondria. Thus, we propose that known differences in cellular calcium loading underlie the discrepant energetic patterns of the two types of VF.


Effect of ischemia and reperfusion on sarcoplasmic reticulum calcium uptake. To investigate the mechanism underlying postischemic cardiac dysfunction (myocardial stunning), contractility and adenine nucleotide metabolism were studied in three groups of isolated perfused rabbit hearts (control, ischemic, and reperfused), whereas Ca2+ uptake by the sarcoplasmic reticulum (SR) was measured in homogenates obtained from them. The hearts were Langendorff-perfused under constant pressure with Krebs-Henseleit solution at 37 degrees C. Global normothermic ischemia was produced by closing the perfusion line. In the reperfused group, after 15 minutes of ischemia, Krebs-Henseleit solution was perfused for 10 minutes. Developed left ventricular pressure (control, 104 +/- 6.3 mm Hg) and left ventricular dP/dt (2,063 +/- 256.6 mm Hg.sec-1) were significantly decreased in reperfused hearts (left ventricular pressure, 78 +/- 5.9 mm Hg; left ventricular dP/dt, 1,339 +/- 216.3 mm Hg.sec-1). Myocardial ATP content (control, 13.6 +/- 0.98 mumol/g dry wt) decreased during ischemia (4.5 +/- 1.23 mumol/g) but was restored to control level on reperfusion (11.8 +/- 0.68 mumol/g). Maximum velocity of Ca2+ uptake by the SR (Vmax) (control, 49.3 +/- 2.54 nmol.min-1 x mg-1) was significantly depressed by ischemia (36.3 +/- 1.94 nmol.min-1 x mg-1) but was restored to the control value after a 10-minute reperfusion (45.3 +/- 0.79 nmol.min-1 x mg-1). Apparent dissociation constant KCa and the Hill coefficient for Ca2+ uptake were not different between control, ischemia, and reperfusion. To test for the possible role of the SR Ca(2+)-release channel in the effect of ischemia and reperfusion, we measured Ca2+ uptake after incubation of homogenates with 610 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)


Reperfusion arrhythmias in isolated perfused pig hearts. Inhomogeneities in extracellular potassium, ST and TQ potentials, and transmembrane action potentials. We recorded direct current electrograms and local [K+]o at multiple sites and transmembrane potentials at selected sites during reperfusion after 5 minutes and 10 minutes of regional ischemia in isolated perfused pig hearts. After 10 minutes of ischemia, the incidence of ventricular fibrillation (VF) was 38%. At 80-90 seconds after reperfusion, [K+]o was 0.8 mM less than in normal tissue in half of the reperfused tissue, especially in the border zone. This was associated with TQ elevation of +4.5 mV and large peaked T waves. The latter was caused by an abrupt decrease of action potential duration in reperfused tissue, leading to a difference of up to 165 msec with normal tissue. Reperfusion VF started with a closely coupled ventricular premature beat. Activation block between reperfused and normal tissue permitted reentrant activation, leading to VF. Pretreatment with ryanodine (10(-6) M) and reperfusion with elevated [K+] (both of which prevent delayed afterdepolarizations) did not prevent closely coupled ventricular premature beats or VF. Five minutes of ischemia never caused VF. K+ depletion and TQ elevation in the reperfused zone was less frequent and smaller (-0.4 mM and 1.8 mV, respectively). Peaked T waves did not occur, and shortening of the action potential duration was less. We conclude that extracellular K+ depletion and marked action potential duration shortening in the reperfused tissue play a role in the genesis of reperfusion VF, which is caused by reentry. The closely coupled ventricular premature beat that initiates reentry is not caused by delayed afterdepolarizations but most likely by intramural reentry.


Culture of renal arteriolar smooth muscle cells. Mitogenic responses to angiotensin II. We cultured smooth muscle cells from rat renal preglomerular arterioles by injecting a suspension of iron oxide into the left ventricle, separating the arterioles magnetically, and growing cells from explants. In passaged cultures we ascertained vascular smooth muscle purity of > 98% by morphology; contraction to norepinephrine and angiotensin; positive immunofluorescence staining through the sixth passage with monoclonal antibodies to smooth muscle-specific alpha- and gamma-isoactins, myosin, and desmin; and the absence of von Willebrand factor. Angiotensin II (10(-12)-10(-5) M) induced dose-dependent DNA synthesis and proliferation of subcultured (three times) arteriolar smooth muscle cells from a growth-arrested state (p < 0.01). Angiotensin II (10(-5) M) also induced the cells to express c-fos mRNA. We find no previous report of culture of smooth muscle cells from renal preglomerular arterioles. Our findings also provide evidence that angiotensin II is mitogenic to arteriolar muscle cells and thus may be involved in their hyperplasia accompanying hypertension.


Relation between vasa recta blood flow and renal interstitial hydrostatic pressure during pressure natriuresis. Pressure natriuresis may be mediated through increases in inner medullary vasa recta blood flow (QVR). By means of acute renal decapsulation to prevent increases in renal interstitial hydrostatic pressure (RIHP), the effect of increases in QVR in the presence and absence of changes in RIHP in the natriuretic and diuretic responses to increases in renal perfusion pressure (RPP) was evaluated. Blood flow in descending (QDVR) and ascending (QAVR) vasa recta was determined in the exposed papilla by fluorescence videomicroscopy in anesthetized euvolemic Munich Wistar rats. In rats with intact renal capsules (n = 12), increases in RPP from 101 +/- 0.5 to 132 +/- 2.9 mm Hg caused significant increases in QDVR (from 4.7 +/- 0.9 to 5.5 +/- 0.9 nl/min, p < 0.001) and QAVR (from 2.8 +/- 0.2 to 3.5 +/- 0.2 nl/min, p < 0.001) in association with increases in RIHP (from 4.6 +/- 1.3 to 7.6 +/- 1.3 mm Hg, p < 0.001), urine flow (from 16.2 +/- 2.6 to 20.2 +/- 3.2 microliters.min-1 x g kidney wt-1, p < 0.01), and urinary sodium excretion (from 2.10 +/- 0.38 to 3.36 +/- 0.62 mu eq.min-1 x g kidney wt-1, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Partial coronary stenosis is sufficient and complete reperfusion is mandatory for preconditioning the canine heart. Repeated brief episodes of total coronary artery occlusion (i.e., severe ischemia), each separated by brief periods of reperfusion, reduce infarct size after a subsequent sustained ischemia. The importance of the intensity of ischemia during these coronary artery occlusions and the role of the following transient reflow are poorly understood. Therefore, our objective was to determine whether moderate preconditioning ischemia induced by partial coronary artery stenosis (reducing coronary flow to approximately 50% of its baseline values), with or without a brief period of total reperfusion, could precondition the canine myocardium. Dogs were randomized to receive one of three preconditioning "treatments": the R(-) group underwent 15 minutes of partial coronary stenosis without subsequent brief reperfusion (n = 8); the R(+) group underwent 15 minutes of partial coronary stenosis followed by 10 minutes of full reflow (n = 8); and the control group underwent no intervention (n = 8). All dogs then underwent 1 hour of total coronary artery occlusion and 4.5 hours of reperfusion. Both treated groups were equally and moderately ischemic during partial stenosis: myocardial blood flow in the inner two thirds of the left ventricular wall averaged 0.25 +/- 0.05 and 0.31 +/- 0.07 ml/min per gram in the R(-) and R(+) groups, respectively (p = NS). Furthermore, all three groups were equally and severely ischemic during sustained total occlusion: myocardial blood flow in the inner two thirds of the left ventricular wall averaged 0.06 +/- 0.05, 0.05 +/- 0.03, and 0.07 +/- 0.03 ml/min/g in control, R(-), and R(+) groups, respectively (p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)


Ejection load changes in aortic stenosis. Observations made after balloon aortic valvuloplasty. To investigate complementarity and competitiveness between the intrinsic and extrinsic components of the total left ventricular systolic load, hemodynamic data from 18 elderly subjects with severe aortic stenosis were analyzed before and after balloon dilation of the stenosed aortic valve. Multisensor micromanometric pressure measurements allowed calculation (simplified Bernoulli equation) of the ejection velocity and aortic input impedance spectra. Despite a 32% increase in the aortic valve area (from 0.56 +/- 0.04 to 0.74 +/- 0.05 cm2 [mean +/- SEM], p < 0.01), the peak left ventricular systolic pressure fell by only 12% (from 189 +/- 10 to 167 +/- 8 mm Hg, p < 0.01). This was accompanied by an increase in the impedance at the same cardiac output. In a subset of patients (n = 9) in whom the peak aortic systolic pressure rose after valvuloplasty (from 115 +/- 10 to 128 +/- 12 mm Hg, p < 0.01), a 40% increase in the aortic valve area was accompanied by a marked increase in the aortic input impedance. In this subset, the steady component of the aortic input impedance increased by 24% (from 960 +/- 96 to 1,188 +/- 134 dyne.sec/ml, p < 0.05), and the characteristic impedance increased by 25% (from 106 +/- 13 to 132 +/- 19 dyne.sec/ml, p < 0.05). Because of an increased aortic impedance acutely following the procedure, the total left ventricular systolic load after balloon dilation of the stenotic valve was only slightly decreased despite a significant increase in aortic valve area. This represents an example of complementarity and competitiveness between the intrinsic and extrinsic components of the total systolic ventricular load. It may explain why improvement in left ventricular performance may be modest acutely following balloon aortic valvuloplasty.


Branching patterns in the porcine coronary arterial tree. Estimation of flow heterogeneity. The aim of this study is to quantify the porcine coronary arterial branching pattern and to use this quantification for the interpretation of flow heterogeneity. Two casts of the coronary arterial tree were made at diastolic arrest and maximal dilation. The relation between length and diameter of arterial segments was quantified, as well as the area expansion ratio and diameter symmetry of vascular nodes. These relations were used to construct computer models of the coronary arterial tree, covering diameters between 10 and 500 microns. Topology of these simulated trees was analyzed using Strahler ordering: Bifurcation ratio, diameter ratio, and length ratio were constant along orders 2-8 and equal to 3.30, 1.51, and 1.63, respectively. In each order, the number of segments per Strahler vessel was almost geometrically distributed. For the lowest orders, these predictions were confirmed by direct observations. From the network model, local pressure and flow were also predicted: Pressure fell from 90 to 32 mm Hg at the 10-microns level. The coefficient of variation (CV) of flow in individual segments was dependent on the number of perfused terminal segments (Nt) according to the fractal relation CV(Nt) approximately Nt(1-D), where D is the fractal dimension (1.20). CV of flow in 1-g tissue units was predicted to be 18%. This study shows that the structure of the coronary arterial bed is an important determinant of the fractal nature of local flow heterogeneity.


Brain death-induced impairment of cardiac contractile performance can be reversed by explantation and may not preclude the use of hearts for transplantation. The shortage of suitable donor hearts for cardiac transplantation is exacerbated by the exclusion of those that exhibit contractile malfunction during the period after brain death but before excision. We have replicated the phenomenon of brain death-induced hemodynamic deterioration in the rat in vivo. After 60 minutes of brain death (defined as the absence of electrical activity in the brain), a variety of indicators of cardiac contractile function fell by approximately 50% (thus cardiac index fell from 21 +/- 2 to 11 +/- 1 ml/min per 100 g body weight). However, once excised and perfused ex vivo, the hearts recovered a level of cardiac function that was identical to that from control animals that had not been subjected to brain death. Similarly, when hearts were excised, stored (6 hours at 4 degrees C), and reperfused ex vivo with blood, they also recovered a functional capability identical to that of normal hearts from animals that had not been subjected to brain death. Our results question whether hemodynamic instability in brain-dead individuals is necessarily an irreversible detrimental cardiac phenomenon and whether these hearts should be excluded from transplantation.


Restoration of cerebrovascular CO2 responsivity by glutamine synthesis inhibition in hyperammonemic rats. Hyperammonemia increases brain glutamine levels, causes astrocytic swelling, and depresses cerebral blood flow (CBF) responsivity to CO2. Methionine sulfoximine (MSO) inhibition of glutamine synthetase activity, known to be enriched in astrocytes, prevents ammonia-induced increases in brain glutamine and water content. We tested the hypothesis that inhibition of glutamine accumulation restores CBF responsivity to CO2 during acute hyperammonemia. Pentobarbital-anesthetized rats treated with either vehicle or MSO (150 mg/kg i.p.) received a 6-hour intravenous infusion of either sodium or ammonium acetate. With subsequent induction of hypercapnia, CBF increased from 113 +/- 14 (mean +/- SEM) to 194 +/- 9 ml/min per 100 g in control rats but was unchanged from 107 +/- 13 to 79 +/- 10 ml/min per 100 g in hyperammonemic rats. Treatment with MSO in hyperammonemic rats restored the CBF response to hypercapnia (from 73 +/- 8 to 141 +/- 14 ml/min per 100 g). With induction of hypocapnia, CBF decreased from 114 +/- 11 to 88 +/- 11 ml/min per 100 g in control rats but increased from 112 +/- 13 to 142 +/- 19 ml/min per 100 g in hyperammonemic rats. Treatment with MSO in hyperammonemic rats did not fully restore the response to hypocapnia but prevented the paradoxical increase in CBF (from 80 +/- 8 to 80 +/- 8 ml/min per 100 g). In control rats, MSO did not affect CO2 responsivity. Treatment with MSO prevented ammonia-induced increases in intracranial pressure. Hyposmotic-induced increases in brain water content and intracranial pressure attenuated the CBF response to hypercapnia but, unlike hyperammonemia, did not attenuate the response to hypocapnia. In contrast to hypercapnia, vasodilation in response to arterial hypotension was intact in hyperammonemic rats. We conclude that the grossly abnormal CBF responsivity to CO2 alterations during hyperammonemia is linked to glutamine accumulation rather than ammonia per se. Cerebral edema secondary to glutamine accumulation may contribute in part to abnormal CBF responses, although other aspects of astrocyte dysfunction are likely to be important.


Cholinergically mediated tachyarrhythmias induced by a single extrastimulus in the isolated canine right atrium. Cholinergic agonists and vagal stimulation potentiate the inducibility of atrial fibrillation. To describe the activation patterns and determine the mechanisms that sustain cholinergic fibrillation, tachyarrhythmias were induced with a single extrastimulus in the isolated Krebs-Henseleit-perfused canine right atrium (n = 11) at increasing concentrations of acetylcholine (from 10(-7.5) to 10(-4.5) M). Bipolar electrograms were recorded from 250 epicardial sites simultaneously during control conditions and during extrastimulation (S1S1, 300 msec; S1S2, effective refractory period+5 msec) with and without acetylcholine. Activation sequence maps were constructed from each recording. Without acetylcholine, no tachyarrhythmias were induced. With increasing concentrations of acetylcholine, the refractory period decreased, and nonsustained (< 2 seconds) rapid repetitive responses were induced. At higher concentrations, a sustained (> 2-minute) fibrillation was induced. Activation sequence maps revealed that the rapid repetitive responses were characterized by multiple reentrant circuits. The number of circuits and wavelets increased in a dose-dependent fashion. However, unexpectedly, this trend did not continue when the tachyarrhythmia became sustained. Instead, the reentry tended to stabilize to a small, single, relatively stable reentrant circuit. In conclusion, the data suggest that, in this model, below a critical level of refractory period (< 95 msec) atrial reentrant circuits, unassociated with anatomic obstacles, can become stable and dominate activation.


Interferon-gamma and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells. Nitric oxide (NO) is an important signal substance in cell-cell communication and can induce relaxation of blood vessels by activating guanylate cyclase in smooth muscle cells (SMCs). NO is synthesized from L-arginine by the enzyme NO synthase, which is present in endothelial cells. It was recently shown that SMCs may themselves produce NO or an NO-related compound. We have studied NO production and its effects on energy metabolism in cultured rat aortic smooth muscle cells. It was observed that the cytokines, interferon-gamma and tumor necrosis factor-alpha, synergistically induced an arginine-dependent production of NO in these cells. This was associated with an inhibition of complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate: ubiquinone oxidoreductase) activities of the mitochondrial respiratory chain, suggesting that NO blocks mitochondrial respiration in these cells. Lactate accumulated in the media of the cells, implying an increased anaerobic glycolysis, but there was no reduction of viability. An NO-dependent inhibition of mitochondrial respiration and a switch to anaerobic glycolysis would reduce energy production of the SMCs. This would in turn reduce the contractile capacity of the cell and might represent another NO-dependent vasodilatory mechanism. It could be of particular importance in inflammation, since cytokines released by inflammatory cells may induce autocrine NO production in SMCs.


[Studies on the strain differences of Schistosoma japonicum in the mainland of China. VII. Genetic variation and differentiation of five isolates]. Electrophoretic techniques have been used to survey five different field-collected isolates (i.e., Anhui, Hubei, Guangxi, Sichuan and Yunnan) of S. japonicum from the mainland of China for genetic variation at 7 isozymes markers representing 9 loci. Polymorphic loci were observed for LDH-1, LDH-2, MDH and PGM, respectively, the proportion of polymorphic loci being 44.4%. Average heterozygosity varied between 0.223 and 0.425, with a mean of 0.332. Nei's genetic distance (D) among the populations of 5 isolates gave values between 0.001 and 0.039 with an average of 0.023, indicating that these isolates are very closely related.


[The activity of tumor necrosis factor in schistosomiasis japonica]. The activity of tumor necrosis factor (TNF) in both the culture supernatant of peripheral blood mononuclear cells (PBMC) and the serum was examined in 20 patients of advanced schistosomiasis at 8th week after pyquiton treatment, and 6 rabbits infected with S. japonicum cercariae at 8th and 16th weeks post-infection and using L929 cell cytotoxic assay. The results showed that the capacity of PBMC to produce TNF in response to LPS from patients with late schistosomiasis was significantly lower than that from normal subjects (P less than 0.01), whereas the activity of TNF in serum was significantly elevated (P less than 0.01). The capacity of PBMC to produce TNF and the TNF activity in serum of infected rabbits showed sharp rise at 8th week post-infection (P less than 0.01), and remained high at 16th week, then returned to normal level 8 weeks after treatment. The persistent rise of TNF in the circulation indicate that TNF might take part in the formation of hepatic fibrosis.


[Study on toxicology of a new anthelmintic "85012"]. "85012", a new anthelmintic containing amidine synthesized in our laboratory, was experimentally effective against Nippostrongylus braziliensis and Ancylostoma caninum. In acute and subacute toxicity test, it was proved lowly toxic. There was no evidence that the drug induced any damages to main organs and tissues of animals. In order to evaluate its potential mutagenicity and teratogenicity, micronucleus test, bone marrow metaphase analysis, CHL cells chromosomal aberration assay, spermatic aberration test as well as teratogenicity test were performed. No mutagenic and teratogenic effects were observed.


[Studies on the effect of immunoenhancing reagent "425" on egg granuloma formation in mice infected with Schistosoma japonicum]. Fourteen--34 days after Schistosoma japonicum-infected mice were injected intraperitoneally with immunoenhancing reagent, Chinese angelica root extract ("425"), the specific antibody (IgG) levels were significantly higher in Group III (362.67 +/- 162.21 - 480.00 +/- 289.45) than the control group (66.67 +/- 39.68 - 245.33 +/- 101.56) in experiment one, and in Group II (488.00 +/- 320.38 - 768.00 +/- 267.38) than the control group (256.00 +/- 189.07 - 394.67 +/- 141.06) in experiment two. The egg granulomatous formation were markedly diminished in the injected mice. The ratios of granulomas vs. eggs' mean diameters of Group III of mice were 5.24 +/- 1.04 - 3.95 +/- 0.77 and those of the control group were 6.59 +/- 1.19 - 5.29 +/- 0.94 in experiment one, those of the group II were 3.75 +/- 0.71 - 4.15 +/- 0.73 and those of the control group were 4.94 +/- 0.81 - 5.36 +/- 0.97 in experiment two. Meantime the egg antigen levels in the injected mice might be lower. This study shows that the control of immunomodulation of granulomatous formation in the hosts injected with immunoenhance reagent "425" can be induced.


[Production and characterization of a murine protective monoclonal antibody against Schistosoma japonicum schistosomula]. Eight murine monoclonal antibodies against surface determinants of Schistosoma japonicum (Chinese mainland strain) schistosomula were generated, of which only one monoclonal IgM antibody (N15D9) gave protection at level ranging from 14 to 39% in experiments of passive transfer or inhibition of infectivity while the others did not exhibit significant levels of passive protection. Further characterization of N15D9 antigen specificity showed that 96 and 14 kDa antigen molecules in cercaria, and 132 and 10 kDa in schistosomula could be recognized by N15D9 in Western blot assay. Furthermore, the 96 and 132 kDa molecules could also be recognized by pooled infected human sera while 14 kDa and 10 kDa only by sera from mice vaccinated with 3-hour schistosomula. The molecules recognizable by N15D9 were surface epitopes repeatedly expressed on cercaria, in vitro 3-hour mechanically transformed schistosomula and 5 day lung-stage schistosomula, as demonstrated by indirect immunofluorescence surface binding assay.


[Generation and immunochemical characterization of an anti-egg monoclonal antibody specific to Schistosoma japonicum]. Monoclonal antibodies (McAbs) were generated from mice immunized with soluble egg antigen (SEA) of Schistosoma japonicum. Five of which were specific to SEA of S. japonicum. The isotype of these McAbs was IgG1. Immunoblotting showed that the approximate molecule weight of the antigens recognized by the McAbs were 22 kDa, 116 kDa and greater than 200 kDa respectively. At least 3 isomorphs of the 22 kDa antigen recognized by 1E1 were found at pI 4.6-6 with 2-D Western blot. Moreover, immunoprecipitation using 125I-labeled S. japonicum SEA demonstrated that only one band corresponding to 30 kDa was recognized by 1E1.


[Localization of the antigen in erythrocytic stages of Plasmodium yoelii by immuno-electron microscopy]. In this study, the antigen recognized by the protective McAb M26-32 in erythrocytic stages of P. yoelii was localized by immuno-electron microscopy with LR Whithe resin embedding and colloidal gold probe cytochemical techniques. The results indicated that the antigen which reacts specifically to McAb M26-32 was mainly localized within the cytoplasm of early and late trophozoites, schizonts and merozoites, being the common antigen of asexual blood stages of the plasmodium. The amount of the antigen was on the increase during the development of trophozoite, while a portion of the antigen might be transported outward by exocytosis of the parasites and then be localized in the cytoplasm of the infected erythrocytes adjacent to the parasites.


[Roles of T cell subsets in the protective immunity of mice against Plasmodium yoelii]. BALB/c mice which had developed protective immunity against Plasmodium yoelii (P. y.) challenge were injected with anti-CD4 or anti-CD8 monoclonal antibody, and were then challenged again with P. yoelli. No impairment of protection was observed. CD8+T cells obtained from spleens of these mice were transferred to BALB/c nude mice and induced partial protection, while transfer of CD4+T cells did not so. In P. yoelli-mouse model, the parasites invaded reticulocytes in the early infection, and the infected reticulocytes could be recognized and attacked by sensitized CD8+T cells. In late infection when P. yoelli also invaded mature erythrocytes, the protective effect of CD8+T cells decreased and the main role of protection was played by antibodies.


[In vitro development of sodium artesunate resistance in Plasmodium falciparum]. Plasmodium falciparum (Lab. culture FCR3 isolate) developed resistance to sodium artesunate after exposure to the drug in vitro. The drug effective concentration which resulted in 50 per cent schizont maturation inhibition (IC50) was 1.6 ng/ml (4.1 nmol/L) before exposure to the drug. After 130 days of discontiguous exposure to sodium artesunate in a stepwise fashion, the sensitivity of the isolate to the drug decreased, with its IC50 3-fold higher than that of the parent isolate. The resistance to artesunate decreased significantly after the resistant line was grown in drug-free medium.


[Study on localization of cystic fluid antigen of Cysticercus cellulosae using McAb-IGSS]. A study was undertaken on the localization of specific combination of McAb against cystic fluid antigen of Cysticercus cellulosae by immunogold silver staining method (IGSS). It was shown that the combination sites of McAb F3 were distinct in the frozen and paraffin sections of Cysticercus cellulosae. The black granules in the bladder walls presented double-layer distribution and those in the scolex folded sites concentrated into pieces. The combination sites of McAb labeled by IGSS against cystic fluid antigen of Cysticercus cellulosae were consistent with the distribution of the cell layers of Cysticercus cellulosae. The sections in the control group had scarce and irregular distribution, which could be differentiated distinctly from those in the experimental group.


[An electrophoretic karyotype of Acanthamoeba polyphaga]. An electrophoretic karyotype of Acanthamoeba polyphaga has been preliminarily analysed by means of pulse field gel electrophoresis (PFGE). Ten chromosomal DNA bands are distinguishable on the gel. Using yeast (Saccharomyces cerevisiae) chromosomal DNA as size standard, we estimate the size of the chromosomes to be between about 200 kilobase pairs (kb) and 2 megabase pairs (mb).


[Comparative analysis of amino acids of metacercaria and larvae of diploid and triploid of Paragonimus westermani]. The diploid type of Paragonimus westermani in Heilongjiang Province and triploid type of P. westermani in Liaoning Province were examined for amino acids by high performance liquid chromatography (HPLC). Eleven kinds of amino-acids of diploid type and 15 kinds of triploid type were found in metacercaria; 15 kinds amino acids of diploid type and 16 kinds of triploid type were detected in larvae. The amino acids were more abundant in larvae than in metacercaria and more in the triploid than in the diploid type. The predominant amino acids were cystine and glutamic acid.


[Transmission electron microscopy of tegument and digestive tract of Schistosoma japonicum during long-term cultivation in vitro]. The article reports the result of the observation on the ultrastructure of the tegument and digestive tract of Schistosoma japonicum schistosomula cultured 130 days in vitro in 841 medium, and the 42-day-old adults from infected animal cultured 23 days in 851 medium by transmission electron microscopy. The results showed that the ultrastructure of the tegument including sensory papillae and flame cells was normal. The matrix seemed to have no vacuolization. The surface of the esophagus was highly rugous. Many laminae and lipid droplets filled the lumen of the digestive tract and the tubules-like depressions from basal lamina were occurred. All these structures seemed to have less variation as compared with the prior authors' data from normal worms. It is considered that the above two media which we cesigned were suitable for the growth and development of the schistosomes due to the tegument and digestive tract maintained their metabolic function in the long-term cultivation in vitro. (Figs 1-10).


[Study on free amino acids and protein in hemolymph of Anopheles stephensi]. The changes in the contents of free amino acids in hemolymph of Anopheles stephensi were determined by automatic amino acid analyzer. The changes in hemolymph protein were determined by ultraviolet absorption method. Free amino acids in hemolymph of infected mosquitoes were compared with those in noninfected mosquitoes. At 4 days after blood meal, 6 kinds of amino acids decreased markedly, and 5 kinds of amino acids increased markedly; at 7 days after blood meal, 4 kinds of amino decreased markedly, while 7 kinds of amino acids increased markedly; at 11 days after acids blood meal, 9 kinds of amino acids decreased markedly, and 4 kinds of amino acids increased remarkably. The protein concentration of infected mosquitoes was higher than that of noninfected ones.


[Studies on negative conversion of schistosome-infected snails in mountainous region of Yunnan]. The monthly cercaria shedding of 90 artificially infected Oncomelania snails was observed under a field condition in mountainous region, Shitoudi Village, Weishan County, Yunnan Province. The results showed that 20% of teh snails shed cercaria monthly, 42.2% could shed irregularly and 37.8% stopped releasing cercariae after several times of shedding. Final direction of the snails showed that none cercaria or sporocyst could be found in part of the snails. A total of 304 naturally infected snails were observed individually every month. Samples were taken each month from the snails which did not shed cercaria, then dissected and examined. The negative conversion rate was calculated in the second, third, fourth, fifth and sixth month after the first shedding, which were 36.1, 50.0, 41.0, 39.8 and 2.6% respectively.


[Changes in T lymphocytes and their subpopulations and IgG in peripheral blood from mice infected with Trichinella spiralis]. Acid alpha-naphthyl esterase (ANAE), a cytoplasmic marker, was used to identify the T lymphocytes and their subpopulations in peripheral blood of mice infected with Trichinella spiralis, and Dot-ELISA was used to identify the serum IgG antibody of the infected mice. The results showed that T lymphocytes increased on the d3 after infection, reaching the peak on d14, and then remained in number greater than normal up to d77. The spotted granular ANAE positive cells (Help T cells, Th) decreased and the scattered granular ANAE positive cells (Suppressor T cells, Ts) increased, leading to a drop of the Th/Ts ratio. The reduction in host immune function during T. spiralis infection might be related to the drop of Th/Ts ratio. At the same time, the serum IgG antibody increased on d7 after infection, reaching the peak on d28, and remained higher up to d140. The results indicated that the host's cellular immunological level was lower while the host's IgG antibody level was higher during T. spiralis infection.


[Analysis of newborn larva of Trichinella spiralis by immunoblot]. Newborn larva (NBL) antigens of Trichinella spiralis were analysed by Immunoblot, and were compared with the adult and muscle larva antigens. The SDS-PAGE patterns of NBL somatic constituents consisted of about 40 polypeptide bands, which were obviously different from those of adult and muscle larva. Immunoblot analysis indicated that immunization with NBL could induce a stage specific immune response. The molecular weight of specific NBL antigens were 129, 120, 89, 87, 79, 74, 72, 64, 58, 43, 40, 38, 34, 32, and 20kDa. But during the natural course of the infection, we could not detect the antibodies of anti-NBL in the host.


[The dynamics of antigen and antibody in Schistosoma japonicum egg granuloma and its relationship with the granuloma response]. Synchronous Schistosoma japonicum egg granuloma in the lung of mouse model and PAP (peroxidas-anti-peroxidase) technique were employed to study the dynamics of antigen and antibody in the egg granuloma of S. japouicum and its relationship with the granuloma formation. The granulomatous response began on the 3.5 day after egg injection and increased to the maximal size at the 4th week. Lymphocyte populations and macrophage comprised an important part of lesions during the time of acute granulomatous response (7-28 day). Using PAP staining, the SEA within egg could be detected at high level on the first day after egg injection and then declined gradually. On the contrary, the SEA around egg was minimal at the first week and increased to the peak at the 4th week, then decreased gradually. No antibody could be detected throughout the experimental period (35 days). The results suggested that 1) the antigen of Schistosoma egg is the essential factor for the granuloma formation. 2) S. japonicum egg granuloma could be formed in unsensitized mouse. 3) The mechanism of egg granuloma formation of S. japonicum is similar to that of S. mansoni.


[Epidemiological trend in late stage of control in Malayan filariasis endemic areas with Anopheles anthropophagus as main vector]. During 1982-1990, a longitudinal observation on prevalence trend of malayan filariasis has been made in endemic areas with An. anthropophagus as the main vector. A total of 22,795 person-times of blood examination were made, and 30,439 An. anthropophagus and 10,061 An. sinensis were dissected respectively. The microfilaraemia rate dropped from 1.0% to 0.14%, and the infection rate of An. anthropophagus decreased from 0.74% to 0.09% in 3 endemic villages, while no positive case or infected vector occurred in 5 villages where microfilaraemia cases were absent since the beginning of the study. In a cross-sectional survey, 855 villages of 17 counties has been monitored for 10 years. Out of 213,934 person-times of blood examination, only 56 were positive, the average microfilaraemia rate being 0.0262%, and 94.64% of the positive had already been detected before 1986. Based on these data, it has been suggested that in endemic areas with An. anthropophagus as main vector, when the microfilaraemia rate dropped to less than 1% after control, there was no indication that the rate would upgrade during the survey period. A decline trend of the transmission of malayan filariasis, therefore, has been exhibited.


[Studies on the strain differences of Schistosoma japonicum in the mainland of China. VIII. Immunoreactions in experimental animals]. Observations on the immunoreactivities toward egg antigens of different experimental animals infected with Schistosoma japonicum were made, which included mouse, rat, hamster, rabbit and rhesus monkey. The cercariae applied for infection were shed by snails collected from 5 different isolates, i.e., Anhui, Hubei, Sichuan, Yunnan and Guangxi in the mainland of China. Sera from the same sort of animals were separately pooled according to the different sources of infection mentioned above. Antibody reactivities were evaluated by COPT, LAT and ELISA. In COPT each serum sample was tested with ova from homologous and heterologous isolates, whereas soluble egg antigen merely prepared from Anhui isolate was used in LAT and ELISA. The level of antibodies detected by homologous or heterologous worm isolate antigens was compared. The results suggested that the positive antibody detection rate might not be influenced by antigens prepared from S. japonicum eggs of different isolates in the mainland of China.


[Morphological and taxonomical studies on anisakidae larvae found in marine fishes of China. II. Gulf of Tong King]. A survey on Anisakidae larvae in 29 species (134 specimens) of marine fishes in the Gulf of Tong King has been carried out. Anisakidae larvae were detected in 15 out of 29 species. The detected specimens were identified as larvae of Anisakis simplex, Hysterothylacium and Pseudoterranova. The parasitization rate of Anisakis simplex larvae, the main pathogen of anisakiasis, in fishes was 30.6% (41/134), while the parasitization rates of Hysterothylacium and Pseudoterranova larvae were comparatively low. Hysterothylacium larvae China type I detected from Muraenesox cinereus and Trichiurus haumela was a new record. Their morphological characteristics were summarized as follows: 1. Length 10.78-14.18 mm, Width 0.25-0.38 mm, the length of the esophagus is 1.14-1.73 mm, intestinal cecum 0.77-1.24 mm and ventricular appendage 6.27-8.40 mm, extending parallelly with the intestine to the last quarter of the larva; 2. Boring tooth was present, but mucron was absent; 3. No genital anlage was observed.


[Oxidative phosphorylation of liver mitochondria in Oncomelania snail]. Oxidative phosphorylation of liver mitochondria in Oncomelania snail was separately detected by using oxygen electrode and spectrophotometer. ADP increased oxidative reaction of liver mitochondria from 0.187 to 0.318 mumol O2/mg protein.20 min. When certain substrates of citric acid cycle were added to liver mitochondria of Oncomelania snail, we found that oxidative phosphorylation increased to 0.353-0.444 mumol O2/mg protein.20 min. ATPase was detected in the liver of Oncomelania snail. The oxidative phosphorylation of mitochondria in Oncomelania snail could be markedly inhibited by DNP and molluscicide bromoacetamide, but the latter didn't show the inhibition of ATPase. (Figs. 1,2).


[Ultrastructure of erythrocytic stage of Plasmodium vivax in humans]. After the merozoite entered the erythrocyte, the membrane debris in the parasitophorous vacuoles of early ring form was passed out through a narrow external aperture in erythrocyte to the exterior. The trophozoite was oval or irregular in shape. Ingestion of host cell cytoplasm occurred cystostomally. The asexual parasite possessed cristate mitochondria and was surrounded by a single-membraned pellicle. The gametocyte possessed cristate mitochondria and was surrounded by two unit membranes. The cytoplasm of mature macrogametocytes contained many ribosomes, mitochondria and osmiophilic bodies and a small nucleus while microgametocytes contained fewer ribosomes, osmiophilic bodies and mitochondria and a large nucleus. Three characteristic morphological alterations were observed within the host cells, that is, small vesicles, cytoplasmic cleft and caveola-vesicle complex. The clefts within the cytoplasm of the host erythrocytes were present in all human malarial parasites. The small vesicles distributed all over the cytoplasm were surrounded by a unit membrane. The caveola-vesicle complex consisted of caveolae was surrounded by small vesicles and probably corresponds to a Schüffner's dot. (Figs. 1-13).


[The blood schizontocidal effects of pyronaridine, amodiaquine, mefloquine and qinghaosu on mice infected with Plasmodium berghei]. The asexual stages of P. berghei ANKA were completely eliminated as revealed in a "4-day suppressive test" with the daily dose of pyronaridine 12.5 mg base/kg or amodiaquine 25 mg base/kg. Mefloquine 25 mg base/kg and qinghaosu 100 mg/kg though exerted obvious suppressive effect, the cure rates were only 50% and 0%, respectively. In treating chloroquine-sensitive P. berghei ANKA strain pyronaridine exhibited the best therapeutic activity, which was followed by amodiaquine, mefloquine and quinghaosu. In treating moderately chloroquine-resistant P. berghei NS line the cure rate of pyronaridine 12.5 mg/kg.d x 4 was 70%, but none of the 10 infected mice from any group was cured by amodiaquine 100 mg/kg.d, mefloquine 100 mg/kg.d or qinghaosu 200 mg/kg.d. Though the latter 3 drugs showed prominent suppressive effects, parasitemia remained positive or recrudesced after dosing. We demonstrate that parasites resistant to chloroquine had cross resistance to amodiaquine, mefloquine and inghaosu at various degrees. Amodiaquine 100 mg/kg.d, mefloquine 100 mg/kg.d and qinghaosu 200 mg/kg.d exhibited no obvious suppressive activity on highly pyronaridine-resistant line of P. berghei, indicating the existence of cross resistance to pyronaridine.


[Observation of ultrastructure and drug sensitivity of in vitro cultured exoerythrocytic form of Plasmodium berghei]. The ultrastructure of in vitro cultured exoerythrocytic stage (EE) of Plasmodium berghei (P. b.) was observed under transmission electron microscope (TEM). The drug sensitivity of EE was also measured in vitro. The EE was cultured in monolayer host cell, fixed and embedded in situ. The ultrathin sections were prepared and examined by routine methods. The TEM pictures showed that the fine structure of in vitro cultured EE was similar with that of EE grown in rat hepatocytes in vivo, as described by Meis et al. The parasite was found within a parasitophorous vacuole, with nucleus, mitochondria, endoplasmic reticula and Golgi apparatus were observed inside the parasite. After 24 h cultivation, the EE was incubated for 48 h in medium containing a serial concentrations of primaquine or chloroquine, then examined under light microscope. The percentage of abnormal parasites was calculated. The preliminary results showed that the sensitivity of cultured P. b. EE to primaquine and chloroquine was significantly different. At the same concentration of 1 x 10(-5) mol/L, the percentage of abnormal parasites was 38.5 +/- 3.9% and 5.7 +/- 1.9%, respectively. These results demonstrated that the in vitro cultivation system of P. b. EE would have potential utility in antimalarial drug research. (Figs. 1-6).


[The first record of human natural infection of Echinochasmus liliputanus]. Human natural infection with Echinochasmus liliputanus was found for the first time from the inhabitants of Hexian county of Anhui Province in Spring, 1991. Sixty worms collected from 3 infected persons were studied morphologically. The adult worms are leaf-shaped, 1519.4-2056.3 x 466.4-564.0 microns in size. The width of the collar is 235.8-297.3 microns. A row of 24 collar spines with size of 22.5-35.6 x 8.8-10.6 microns is arranged on the collar symmetrically with dorsal and ventral interruption. The oral sucker is terminal, 107.6-148.6 x 102.5-148.6 microns. The length of the prepharynx is 25.6-66.7 microns, the pharynx, 97.3-127.8 microns and the oesophagus 117.9-205.0 microns. The caeca 764.3-1,248.8 x 21.0-39.0 microns extends to the distal end of the body. The acetabulum is anterior to the middle level of the body, 205.0-240.9 x 205.0-235.8 microns in size. Two testes, situated in the posterior one third of the body, is slightly oval, in tandem; the anterior one is 133.3-199.9 x 199.9-256.4 microns in size, and the posterior one, 178.9-251.3 x 164.0-246.0 microns. The Cirrus pouch is kidney-shaped, 211.5-248.5 x 112.8-194.8 microns in size, located between the bifurcation of the intestine and the acetabulum, containing the seminal vesicle and cirrus. The ovary is oval in shape, 71.8-92.3 x 76.8-97.4 microns in size, situated in the middle of the body. The vitellaria are distributed on each side from the acetabulum to subterminal, consisting of many follicles. The uterus is short, convoluted between the anterior testis and the acetabulum, containing 0-6 eggs.(ABSTRACT TRUNCATED AT 250 WORDS)


[Preparation and identification of anti-Trichomonas vaginalis monoclonal antibodies]. Hybridomas producing monoclonal antibodies (McAb) directed against Trichomonas vaginalis have been produced by fusing NSI myeloma cells with spleen cells of BALB/c mice immunized with Trichomonas vaginalis. IFA technique was used to test the binding activity of four McAbs produced. The McAb belonged to the IgG subtypes IgG1 (2A2, 2A4 McAb), IgG3 (2H9 McAb) and IgG 2b (2A12 McAb). Three McAbs, designated 2A2, 2A4, 2A12, reacted with a surface membrane component of live Trichomonas vaginalis. One (2A12) of them produced complement-dependent cytolysis of the parasites. Others (2A2,2A4) produced complement-independent cytotoxicity of the parasites. 2H9 McAb which reacted with the nucleus of the organisms did not agglutinate the parasites. The four McAbs which did not have cross reaction with some protozoa of Zoomastigophorea species were specific antibodies against Trichomonas vaginalis. (Figs. 1-3).


[Isolation of germinal cells from the secondary cysts of Echinococcus granulosus harbored in mice]. The secondary cyst tissues derived from mice infected with protoscoleces of Echinococcus granulosus for 8-10 months were digested with 0.25% trypsin at 37 degrees C for 30 min. The separation of different cells in the remaining suspension was achieved by discontinuous gradient centrifugation. The germinal cells were washed 3 times with ice-cold HBSS, and then cultivated in the medium of RPMI 1640 supplemented with 20% of calf serum. The cells were kept in an incubator at 37 degrees C in an atmosphere of 95% air-5% CO2. After incubation for 5-7 days, the germinal cells began to multiply accompanied by the enlargement of cells as compared with those before incubation. The surface of both isolated and/or cultured cells showed smooth appearance examined by scanning electron microscopy. Immunofluorescence assay and enzyme-linked immunosorbent assay had been used for examining the specific antigenicity of the cells. The results showed that antigen components of E. granulosus were detected either on cell surface or in soluble proteins of the cells. Furthermore, 120 NIH female mice were inoculated intraperitoneally with 1-5 x 10(7) cultured germinal cells and sacrificed 1-3 months after inoculation. Only 2 cystic materials had been detected in two mice. Of which, one located in the liver and the other in peritoneal cavity of the animals. Histological examination noted that the cystic materials consisted of germinal layer and cyst fluid, but no laminated layer was observed. The above mentioned evidence demonstrated that the cells isolated from the cysts of E. granulosus were germinal cells.(ABSTRACT TRUNCATED AT 250 WORDS)


[Chemical synthesis and cloning of Plasmodium falciparum 45 peptide antigen gene]. We have synthesized a 162 bp gene of human Plasmodium falciparum hybrid peptide antigen by the solid-phase phosphoramidite method with ABI 381A DNA synthesizer. The gene encodes three fragments of the relative molecules 83 kDa, 55 kDa and 35 kDa merozoite-specific proteins and two CS repeats or four peptides. The gene with the designed two cohesive ends was divided into 8 fragments to be synthesized. All synthetic fragments were annealed and ligated with T4 DNA ligase to form double DNA chain. This synthetic gene was recombined with P-Blue script as vector and transformed into E. coli JM109. The positive recombinants were screened out by dot hybridization and enzyme analysis. The DNA sequence analysis showed that the synthesized human Plasmodium falciparum hybrid peptide antigen gene was identical with the designed one. (Figs. 1-4).


[DNA sequence of surface antigen gene gp195 of two Chinese isolates of Plasmodium falciparum from Hainan]. Amplification of DNA sequence of surface antigen gene, block II of gp195 from 2 Chinese isolates of Plasmodium falciparum FCC 7801/HN B3 and FCC M21/HN from Hainan, China, was established by using the polymerase chain reaction. A comparison of the 154 peptide sequences of the 2 Chinese isolates showed that they were identical, but there was a difference of 10 peptides among the established 154 peptides between the two Chinese isolates and MAD 20 (a Papua New Guinea isolate). The need to determine the fragments having protective effect against malarial infection, and to study the genetic basis of the antigen polymorphism of Chinese strains of Plasmodium falciparum is evident.


[Treatment of soil-transmitted helminth infections by anthelmintics in current use]. The efficacy of broad-spectrum anthelmintics in current use was studied in Hengshan County, Hunan Province. The vermicides under study include albendazole (400mg, single dose), mebendazole composite (mebendazole 100 mg and levamisole 25mg bid x 3d), oxantel pyrantel pamoate composite (pyrantel pamoate 150 mg and oxantel pamoate 150 mg bid x 2d), and pyrantel pamoate composite (base 10 mg/kg, single dose). Therapeutic effect assessed 2 weeks after medication revealed Ascaris egg negative rates or cure rates (CR) of 97.5-100% for the former 3 regimens, and 80.9% for the latter one; while CR for hookworm infection were 95.4%, 78.6-100%, 96.7% and 83.3%, respectively. A follow-up survey pursued 4 weeks post treatment showed no significant difference in CR for the above regimens. Judging from CR in Trichuris trichiura infection, pyrantel pamoate composite was recommended as the drug of choice (89.3%), which was followed by mebendazole composite (64.6-83.8%) and albendazole (28.2-42.6%), whereas pyrantel pamoate was inefficacious. Obvious egg reduction rates were evidenced post application of the above drugs in trichuriasis treatment except pyrantel pamoate at single dose.


Neurohormonal inhibition and hemodynamic unloading during prolonged inhibition of ANF degradation in patients with severe chronic heart failure. The purpose of this study was to investigate the therapeutic potential of prolonged inhibition of atrial natriuretic factor (ANF) degradation in patients with severe chronic heart failure. The effects of repeated doses of the endopeptidase inhibitor candoxatrilat (150 mg i.v.) were examined over a 24-hour period in patients with severe chronic heart failure (New York Heart Association class III-IV). Plasma alpha-hANF(99-126) was elevated at baseline (235 +/- 59 pg/ml), increased 2.5-fold at 2 hours after the first dose, and remained significantly elevated throughout the 24-hour protocol. In contrast, pro-hANF(31-67) decreased from 3,151 +/- 616 to 2,072 +/- 362 pg/ml (p less than 0.05). Cardiac index (CI) increased only transiently after the first dose of candoxatrilat (CI, 2.11 +/- 0.2 to 2.67 +/- 0.28 l/min/m2, p less than 0.05). Sodium excretion increased sixfold (p less than 0.05) 2 hours after the first dose of candoxatrilat and remained significantly elevated throughout the protocol. Degree of natriuresis and diuresis in response to candoxatrilat was closely related to baseline cardiac output. Glomerular filtration rate and volume excretion did not change significantly. Pulmonary capillary wedge pressure fell from 23 +/- 3 to 18 +/- 3 mm Hg (p less than 0.05) and remained below baseline throughout the 24 hours. Arterial pressure, heart rate, and total peripheral resistance did not change significantly during the 24-hour period. Urinary cGMP excretion increased fivefold (p less than 0.05), whereas urinary ANF immunoreactivity and plasma cGMP levels remained unchanged. Excretion of prostacyclin metabolite 6-keto-PGF-1 alpha increased 3.3-fold (p less than 0.05). Plasma norepinephrine and epinephrine levels decreased significantly after candoxatrilat and remained suppressed over the 24-hour period. There was also a transient reduction in plasma vasopressin, aldosterone levels, and plasma renin activity. Hematocrit, total protein content, and plasma albumin concentrations did not change, indicating that no fluid shift into the extravascular space had occurred. 1) The inhibition of ANF degradation causes sustained drop in left and right atrial pressures that appears to be mediated by an inhibition of neurohumoral activity; 2) concomitant inhibition of bradykinin breakdown (which in turn stimulates renal prostacyclin synthesis) contributes to natriuresis; 3) the close correlation between renal response and baseline cardiac index indicates that an inadequate renal perfusion secondary to low cardiac output diminishes the efficacy of this treatment modality. This spectrum of action would be advantageous for a first-line diuretic agent early in the course of disease rather than in patients with advanced chronic heart failure.


Sex-associated differences in left ventricular function in aortic stenosis of the elderly. In aortic stenosis, the response of the left ventricle to pressure overload varies from compensated hypertrophy to overt heart failure. The determinants of left ventricular adaptation are poorly understood. Left ventricular function was compared to assess the role of sex in 34 women and 29 men 60 years or older with both hemodynamic and echocardiographic data characteristic of severe aortic stenosis and no important coronary artery disease. Despite a similar degree of left ventricular outflow obstruction in women versus men (aortic valve area 0.54 +/- 0.20 versus 0.59 +/- 0.19 cm2, NS), the left ventricle of women had a greater fractional shortening (37 +/- 12 versus 25 +/- 12%, p = 0.001), achieved a smaller end-systolic chamber size (1.82 +/- 0.64 versus 2.17 +/- 0.65 cm/m2, p = 0.04), and generated more pressure (210 +/- 35 versus 182 +/- 29 mm Hg, p = 0.001) with a greater maximum positive dP/dt (2.153 +/- 794 versus 1,595 +/- 384 mm Hg/sec, p = 0.02). The men had a lower cardiac index (2.12 +/- 0.59 versus 2.49 +/- 0.63 l/min/m2, p = 0.02), higher mean pulmonary artery pressure (35 +/- 13 versus 27 +/- 10 mm Hg, p = 0.01), and shorter ejection period (340 +/- 40 versus 370 +/- 40 msec, p = 0.02). Women and men were equally symptomatic. Supernormal left ventricular ejection performance was present in 41% of the women and only 14% of the men (p = 0.002). This subgroup of women had a small, thick-walled chamber (end-diastolic radius to thickness ratio, 1.58 +/- 0.52 versus 2.45 +/- 0.51 in control women, p = 0.01) with low end-systolic wall stress. Subnormal ejection performance was present in 64% of the men and only 18% of the women (p = 0.002). This subgroup of men had an increased chamber size and high end-systolic wall stress compared with control men. Greater left ventricular mass was present in men compared with women (211 +/- 55 versus 179 +/- 55 g/m2, p = 0.03). Sex is a factor in left ventricular adaptation to valvular aortic stenosis in adults 60 years or older.