Transient mitral regurgitation following vagal stimulation-induced ventricular arrhythmia in rabbits: a color Doppler echocardiographic study.

BACKGROUND AND AIM OF THE STUDY: Cervical vagal stimulation in rabbits frequently causes systolic murmur with bigeminy due to premature ventricular contractions. The bigeminy disappears in a few minutes, but the systolic murmur persists for a few days. Peculiar lesions of the mitral valves, mitral annulus and papillary muscles, and an increase in left atrial weight, frequently develop in a week. In this study, color Doppler echocardiography was used to examine whether the systolic murmur was due to mitral regurgitation. METHODS: Echocardiographic monitoring was carried out in anesthetized rabbits restrained in the supine position. RESULTS: Doppler echocardiography and phonocardiography showed systolic murmur at 6 h, three days, and at one, two, three and four weeks after vagal stimulation. At 6 h after stimulation, phonocardiography showed systolic click and late systolic murmur; Doppler echocardiography showed marked mitral regurgitation. The systolic murmur and mitral regurgitation were attenuated and the papillary muscle was swollen three days after vagal stimulation. Following stimulation, mitral regurgitation disappeared within one week, and papillary muscle swelling improved after three weeks. CONCLUSION: Doppler echocardiography confirmed that systolic murmur caused by vagal stimulation in rabbits was due to mitral regurgitation.

Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme.

Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3'-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.

A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells.

Wild-type and several mutant human manganese superoxide dismutases (Mn-SODs) were produced in a baculovirus/insect cell system and characterized. The enzymatic activity of a homogenate of Sf21 cells, infected with baculovirus carrying wild-type Mn-SOD and grown in the conventional medium, was indistinguishable from that of control cells, but was augmented by supplementation with Mn2+. The protein produced was largely imported into the mitochondria, as judged from the enrichment in the mitochondrial fraction, the mobility of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results of N-terminal processing, which was confirmed by sequencing of the purified enzyme. However, a significant amount of precursor was also detected by an antibody raised against the human Mn-SOD signal peptide. While both Mn2+ and Fe3+ stimulated Mn-SOD accumulation within mitochondria, the active form was produced in the presence of submillimolar Mn2+ only. Amino acid substitutions at a signal peptide-cleavage site, His-Ser-Leu4 to Pro-Met-Va14, in the mature Mn-SOD prevented the processing of the precursor protein, and thus resulted in the accumulation of the precursor protein within mitochondria, as judged on immunostaining with an anti-Mn-SOD antibody. Mutant Mn-SODs with a truncated signal peptide or carboxyl region (8, 13, and 42 amino acid residues in the mature form) were barely solubilized, even with a nonionic detergent, and exhibited no activity, suggesting inappropriate folding of these mutant SODs. They were also susceptible to proteolytic degradation, while the wild-type and precursor forms were resistant. Thus, the baculovirus/insect cell expression system appears to be adequate for the analysis of mitochondrial import using intact cells as well as for the large scale production of active Mn-SOD.

Legionella infection and control in occupational and environmental health.

This review describes the microbiological characteristics of Legionella species, their habits in the environment, source and route of infection, pathogenesis, symptoms and treatment of legionellosis, and disease outbreaks worldwide. We also describe prevention measures to deal with this organism in the work environment. Particularly in Japan, the present measures and countermeasures against legionellosis are inadequate when compared with those Europe and the United States because occupational and environmental medicine in Japan has not been structured from a microbiological viewpoint. As a result, workplace inspections have not covered cooling towers. We surveyed the cooling towers provided in work environments and in hospitals in Kitakyushu City for contamination by Legionella species. The surveillance definitely revealed that we are surrounded by cooling towers contaminated with Legionella. We conclude that in Japan, occupational and environmental physicians should routinely monitor the water in cooling towers.

Instability of mutant Cu/Zn superoxide dismutase (Ala4Thr) associated with familial amyotrophic lateral sclerosis.

In about 20-25% of cases of familial amyotrophic lateral sclerosis (FALS) patients have mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The mechanism through which the mutations in the SOD1 gene cause ALS still remain unknown. We performed pulse-chase experiments using a system for the transient expression of human SOD1 in COS7 cells to examine whether the Ala4Thr mutation, which we previously reported, decreases the stability of SOD1. The expression vector (pEF-BOS) carrying the wild-type or mutant (Ala4Thr) human SOD1 cDNA was transfected into COS7 cells, and transiently expressed human SOD1 was then metabolically radiolabeled. Half-lives of the wild-type and the Ala4Thr mutant SOD1 were determined to be 78 h and 18 h, respectively. These results suggest that the Ala4Thr mutation in SOD1 decreases the stability of SOD1 and that this instability may play an important role in the pathogenesis of the degeneration of motor neurons in FALS.

The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species.

Selenium-dependent glutathione peroxidase (GPx) plays a protective role in oxidative stress-induced apoptosis. In this study, we demonstrated that MDBK cells, a bovine renal epithelial cell line, exhibited internucleosomal DNA fragmentation characteristic of apoptotic cell death under selenium-deficient conditions with lower doses of hydrogen peroxide (H2O2) than under selenium-supplemented ones. This was due to a decreased amount of GPx in the cells under selenium-deficient conditions, because other antioxidative enzyme activities were not affected by the selenium supplementation. Cumene hydroperoxide also induced DNA fragmentation in selenium-deficient cells but no ladder formation was observed. Flow cytometric analysis showed that selenium-deficient cells were less capable of scavenging intracellular peroxides after exposure to exogenous H2O2 than selenium-supplemented ones. In contrast, there was no difference in viability between selenium-supplemented and non-supplemented cells in cell survival after exposure to menadione, which activates the electron transport system and increases intracellular superoxide radicals. Clofibrate, a peroxisomal proliferator and an inducer of catalase (CAT), partially protected both Se-deficient and Se-supplemented cells from exogenous H202. We concluded that selenium-deficient cells were more easily brought to apoptotic cell death by peroxides, but not by superoxide radicals, than selenium-supplemented ones and that CAT could compensate for the depletion of GPx to a certain degree by scavenging H2O2.

[Legionella infection in occupational and environmental health].

Air condition systems are indispensable for amenity in the work environment. It is known that Legionella species are widely distributed in the water of cooling towers, and that the bacteria are responsible for community-acquired pneumonia (Legionnaires' disease) and for influenza-like symptoms (Pontiac fever). Furthermore, Legionella species are associated with building-related illness. In Japan, however, prevention and countermeasures are inadequate against legionellosis compared to those of Europe and the USA. This is because occupational and environmental medicine in Japan has not been based on a microbiological point of view, and that workplace inspections have not covered cooling towers. Therefore, Legionella species in the water of coolig towers have not been routinely monitored in the work environment. This review describes the microbiological characteristics of Legionella species, their habits in the environment, the source and route of infection, the pathogenesis, the symptoms and treatment of legionellosis, outbreaks of this disease throughout the world, and how to deal with this organism in the work environment to prevent legionellosis.

Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells.

We describe the use of a baculovirus expression system to overproduce human Cu,Zn-superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA synthesized a large amount of Cu,Zn-SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose-dependent manner when Cu2+ and Zn2+ were added to the medium. Cells grown in media supplemented with Cu2+ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu2+ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti-human Cu,Zn-SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly41Asp, His43Arg, and Gly85Arg exhibited 47, 66, and 99% of wild-type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.

Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells.

We have cloned cDNAs encoding endothelial nitric oxide synthase (ecNOS) from a human fetal liver cDNA library. Overproduction of ecNOS in a baculovirus/insect cell expression system in conventional medium yielded a large amount of ecNOS protein localized in particulate components, but ecNOS activity was low. This activity was increased by addition of hemin to 2.5-fold. While a precursor for heme biosynthesis increased the activity, inhibitors of heme biosynthesis reduced the ecNOS activity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chromatography. The resultant ecNOS required supplementation with cofactors for activity, but it did not require hemin. Binding of a protoporphyrin IX heme was confirmed by a pyridine hemochrome assay.

Nitric oxide synthase from rat colorectum: purification, peptide sequencing, partial PCR cloning, and immunohistochemistry.

Nitric oxide synthase (NOS) has been purified over 6,500-fold with a 3.4% yield from rat colorectum with 2',5'-ADP-Sepharose, DEAE cellulose, and gel filtration. The purified enzyme gave a single band corresponding to an apparent molecular mass of 160 Dka on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When assayed in the requisite presence of L-arginine, CaCl2, NADPH, calmodulin, tetrahydro-L-biopterin, and FAD, the purified enzyme exhibited a specific activity of 328 nmol/min/mg L-citrulline formed and an apparent Km for L-arginine of 2.9 microM. Amino acid sequencing of 12 peptides revealed identical sequences to that of the neuronal type enzyme except for two altered amino acid residues. When partial reverse transcription-polymerase chain reaction of RNA from rat colorectum and cerebellum was performed using primers designed according to the amino acid sequences determined, these amino acid changes were found in both cDNA fragments, indicating the identity of the colorectal enzyme to the cerebellar one. A polyclonal antibody raised against NOS purified from rat cerebellum cross-reacted with the NOS from colorectum but not that from IFN-gamma stimulated macrophage-derived cells, RAW 264.7. Immunohistochemical analysis of the colorectum using this specific antibody indicated that Auerbach's plexus is strongly immunoreactive, supporting the hypothesis that NO is an inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum.

Chromosome mapping of five human cardiac and skeletal muscle sarcoplasmic reticulum protein genes.

Fluorescence in situ hybridization (FISH) experiments were performed using genomic and complementary DNA probes in order to determine the location on human chromosomes for five genes expressed in cardiac and skeletal muscle sarcoplasmic reticulum. The chromosome location of each gene was determined in terms of both cytogenetic bands and fractional chromosome length. The ATP2A2 gene, expressing the SERCA2 isoform of the Ca2+ pump, maps to bands 12q23-q24.1, the phospholamban gene (PLN) to 6q22.1, the human skeletal muscle calsequestrin gene (CASQ1) to band 1q21, the cardiac calsequestrin gene (CASQ2) to bands 1p11-p13.3, and the cardiac calcium release channel gene (RYR2) to the interval between band 1q42.1 (distal) and band 1q43 (proximal).

Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially lethal condition in which sustained muscle contracture, with attendant hypercatabolic reactions and elevation in body temperature, are triggered by commonly used inhalational anaesthetics and skeletal muscle relaxants. In humans, the trait is usually inherited in an autosomal dominant fashion, but in halothane-sensitive pigs with a similar phenotype, inheritance of the disease is autosomal recessive or co-dominant. A simple and accurate non-invasive test for the gene is not available and predisposition to the disease is currently determined through a halothane- and/or caffeine-induced contracture test on a skeletal muscle biopsy. Because Ca2+ is the chief regulator of muscle contraction and metabolism, the primary defect in MH is believed to lie in Ca2+ regulation. Indeed, several studies indicate a defect in the Ca2+ release channel of the sarcoplasmic reticulum, making it a prime candidate for the altered gene product in predisposed individuals. We have recently cloned complementary DNA and genomic DNA encoding the human ryanodine receptor (the Ca2(+)-release channel of the sarcoplasmic reticulum) and mapped the ryanodine receptor gene (RYR) to region q13.1 of human chromosome 19 (ref. 14), in close proximity to genetic markers that have been shown to map near the MH susceptibility locus in humans and the halothane-sensitive gene in pigs. As a more definitive test of whether the RYR gene is a candidate gene for the human MH phenotype, we have carried out a linkage study with MH families to determine whether the MH phenotype segregates with chromosome 19q markers, including markers in the RYR gene. Co-segregation of MH with RYR markers, resulting in a lod score of 4.20 at a linkage distance of zero centimorgans, indicates that MH is likely to be caused by mutations in the RYR gene.

[A clinical study of amino acid metabolism in cancerous hypoproteinemia--the role of branched chain amino acids (BCAA) and appropriate compositions of BCAA in parenteral nutrition].

In order to determine the nutritional effects of BCAA compositions in the treatment of cancerous hypoproteinemia, the appropriate ratio of I-leu: Leu: Val and the proportion of BCAA to Total Amino Acids were investigated. As for results, indices such as the serum albumin, the RBP and N-balance quickly recovered to normal levels when the ratio of I-leu: Leu: Val was 1.0:1.8:1.0 and the proportion o BCAA to TAA was 31%. These composition thus may be suitable for the treatment of cancerous hypoproteinemia.

Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban.

Complementary DNA (cDNA) clones specific for phospholamban of sarcoplasmic reticulum membranes have been isolated from a canine cardiac cDNA library. The amino acid sequence deduced from the cDNA sequence indicates that phospholamban consists of 52 amino acid residues and lacks an amino-terminal signal sequence. The protein has an inferred mol wt 6,080 that is in agreement with its apparent monomeric mol wt 6,000, estimated previously by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholamban contains two distinct domains, a hydrophilic region at the amino terminus (domain I) and a hydrophobic region at the carboxy terminus (domain II). We propose that domain I is localized at the cytoplasmic surface and offers phosphorylatable sites whereas domain II is anchored into the sarcoplasmic reticulum membrane.

Characterization of recombinant cDNA clones for canine cardiac phospholamban.

Complementary DNA clones specific for phospholamban have been isolated from a canine cardiac cDNA library. The amino acid sequence deduced from the cDNA sequence showed that phospholamban consisted of 52 amino acid residues and was synthesized without an amino-terminal signal sequence. The RNA blot analysis revealed that phospholamban mRNAs were represented by two main species of approximately 1.2kb and approximately 2.8kb. These mRNAs appeared to differ primarily in the length of the 3' untranslated region.

Norepinephrine turnover in the cardiovascular tissues and brain stem of the rabbit during development of one-kidney and two-kidney Goldblatt hypertension.

To assess the role of the sympathetic and central noradrenergic neurons in one- and two- kidney Goldblatt hypertension, we examined the concentration and turnover of norepinephrine (NE) in the aorta, mesenteric artery, left ventricle, hypothalamus, midbrain, and pons medulla of hypertensive and control rabbits. Animals were made hypertensive by constriction of the left renal artery after right nephrectomy (1KGH group) or with the right kidney left intact (2KGH group), or were sham-operated on the renal artery (1KGC and 2KGC groups). At 14 days after the constriction, the blood pressure was increased to 136 +/- 3 mm Hg in the 1KGH vs 98 +/- 3 mm Hg in the 1KGC (p less than 0.001), and 136 +/- 2 mm Hg in the 2KGH vs 94 +/- 2 mm Hg in the 2KGC group (p less than 0.001). Turnover time in the aorta, mesenteric artery, and left ventricle in the 1KGH group was decreased to 47%, 45%, and 65% of that in the 1KGC group, respectively. Results suggest that enhanced sympathetic neuron activity in the cardiovascular system, especially in the arteries, contributes to the development of one-kidney Goldblatt hypertension. Norepinephrine turnover in the cardiovascular tissues in the 2KGH group and in the brain stem in the 1KGH and 2KGH group was not different from that in the control group.

The content and turnover of norepinephrine in the rabbit brain regions and cardiovascular tissues.

The content and turnover of norepinephrine (NE) in the hypothalamus, midbrain, pons-medulla, mesenteric artery, aorta and left ventricle were studied in the normal rabbit. Turnover rate of NE was determined by measuring the rate of decline in NE content of the tissue after blockade of synthesis with alpha-methyl-p-tyrosine. The NE content of the hypothalamus (1.44+/-0.03 microgram/g) was significantly higher than those of the midbrain (0.25+/-0.01 microgram/g) and the pons-medulla (0.36+/-0.01 microgram/g) (p less than 0.001), whereas the rate constant of NE turnover for the pons-medulla (0.213+/-0.009 hr-1) was significantly greater than those for the hypothalamus (0.164+/-0.008 hr-1, p less than 0.001) and the midbrain (0.180+/-0.008 hr-1, p less than 0.01). In the cardiovascular tissues examined, the NE content was highest in the mesenteric artery (6.33+/-0.19 microgram/g), moderate in the left ventricle (2.08+/-0.10 microgram/g) and lowest in the aorta (0.70+/-0.06 microgram/g). The differences among them were significant (p less than 0.001). However, the rate constant of NE turnover for the aorta (0.119+/-0.014 hr-1) was significantly greater than those for the mesenteric artery (0.059+/-0.008 hr-1, p less than 0.001) and the left ventricle (0.069+/-0.006 hr-1, p less than 0.005). The turnover rate of NE in the mesenteric artery was high, 0.372 microgram/g-hr, which suggests the very active NE synthesis. These results indicate that there are regional differences in content and turnover of NE of the cardiovascular tissues as well as of the brain.