Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

Apelin increases atrial conduction velocity, refractoriness, and prevents inducibility of atrial fibrillation.

Previous studies have shown an association between elevated atrial NADPH-dependent oxidative stress and decreased plasma apelin in patients with atrial fibrillation (AF), though the basis for this relationship is unclear. In the current study, RT-PCR and immunofluorescence studies of human right atrial appendages (RAAs) showed expression of the apelin receptor, APJ, and reduced apelin content in the atria, but not in plasma, of patients with AF versus normal sinus rhythm. Disruption of the apelin gene in mice increased (2.4-fold) NADPH-stimulated superoxide levels and slowed atrial conduction velocities in optical mapping of a Langendorff-perfused isolated heart model, suggesting that apelin levels may influence AF vulnerability. Indeed, in mice with increased AF vulnerability (induced by chronic intense exercise), apelin administration reduced the incidence and duration of induced atrial arrhythmias in association with prolonged atrial refractory periods. Moreover, apelin decreased AF induction in isolated atria from exercised mice while accelerating conduction velocity and increasing action potential durations. At the cellular level, these changes were associated with increased atrial cardiomyocyte sodium currents. These findings support the conclusion that reduced atrial apelin is maladaptive in fibrillating human atrial myocardium and that increasing apelin bioavailability may be a worthwhile therapeutic strategy for treating and preventing AF.

Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-ß antibody, angiotensin II (AngII), anti-TGF-ß antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-ß dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-ß dependent.

Degradation of histone deacetylase 4 via the TLR4/JAK/STAT1 signaling pathway promotes the acetylation of high mobility group box 1 (HMGB1) in lipopolysaccharide-activated macrophages.

High mobility group box 1 (HMGB1) has been proposed as crucial in the pathogenesis of many diseases including sepsis. Acetylation of HMGB1 prevents its entry into the nucleus and leads to its secretion from the cell where it can trigger inflammation. We hypothesized that histone deacetylase 4 (HDAC4) controls the acetylation of HMGB1 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells via the janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. The results showed that LPS treatment promoted the degradation of HDAC4 in a proteasome-dependent manner, which led to HMGB1 acetylation. In LPS-activated RAW264.7 cells, treatment with TAK-242 (a toll like receptor 4 inhibitor) and pyridone 6 (a JAK inhibitor) significantly inhibited HDAC4 degradation and acetylation of HMGB1, and thus prevented secretion of HMGB1. Decreased phosphorylation of STAT1 was also observed. Interestingly, HDAC4 overexpression significantly prevented the acetylation and secretion of HMGB1 in both RAW264.7 cells and isolated murine peritoneal macrophages. We conclude that HDAC4 might be a useful target for the treatment of sepsis.

Size-dependent isolation of primordial germ cells from avian species.

Primordial germ cells (PGCs), the precursors of sperm or ova, could be used to generate transgenic animals and interspecies germ-line chimeras, which would facilitate the recovery of endangered species by making their access and manipulation in vitro easier. During early embryogenesis in avian species, PGCs are transported via the bloodstream to the gonadal anlagen. PGCs of most avian species-particularly wild or endangered birds-are not readily isolated from the embryonic bloodstream because germ-cell markers have not yet been defined for them. Here, we report a rapid, efficient, and convenient method for PGC isolation from various avian species. Blood PGCs were isolated based on the difference in size between PGCs and other blood cells, using a microporous membrane. The efficiency of this size-dependent isolation for the White Leghorn chicken was not significantly different from that of magnetic-activated cell sorting, and the isolated cells expressed chicken PGC-related genes and PGC-specific markers. The utility of the method was then verified in Japanese quail (Coturnix japonica), Mallard duck (Anas platyrhynchos), and Muscovy duck (Cairina moschata). Immunocytochemistry and an in vivo migration assay indicated that this method was able to enrich for true embryonic blood PGCs without specific antibodies, and could be applied to the development of avian interspecies chimeras for restoration of wild or endangered species.

Long-term outcomes of septal reduction for obstructive hypertrophic cardiomyopathy.

BACKGROUND: Surgical myectomy and alcohol septal ablation (ASA) aim to decrease left ventricular outflow tract (LVOT) gradient in hypertrophic cardiomyopathy (HCM). Outcome of myectomy beyond 10 years has rarely been described. We describe 20 years of follow-up of surgical myectomy and 5 years of follow-up for ASA performed for obstructive HCM. METHODS: We studied 171 patients who underwent myectomy for symptomatic LVOT obstruction between 1972 and 2006. In addition, we studied 52 patients who underwent ASA for the same indication and who declined surgery. Follow-up of New York Heart Association (NYHA) functional class, echocardiographic data, and vital status were obtained from patient records. Mortality rates were compared with expected mortality rates of age- and sex-matched populations. RESULTS: Surgical myectomy improved NYHA class (2.74±0.65 to 1.54±0.74, p<0.001), reduced resting gradient (67.4±43.4mmHg to 11.2±16.4mmHg, p<0.001), and inducible LVOT gradient (98.1±34.7mmHg to 33.6±34.9mmHg, p<0.001). Similarly, ASA improved functional class (2.99±0.35 to 1.5±0.74, p<0.001), resting gradient (67.1±26.9mmHg to 23.9±29.4mmHg, p<0.001) and provoked gradient (104.4±34.9mmHg to 35.5±38.6mmHg, p<0.001). Survival after myectomy at 5, 10, 15, and 20 years of follow-up was 92.9%, 81.1%, 68.9%, and 47.5%, respectively. Of note, long-term survival after myectomy was lower than for the general population [standardized mortality ratio (SMR)=1.40, p<0.005], but still compared favorably with historical data from non-operated HCM patients. Survival after ASA at 2 and 5 years was 97.8% and 94.7%, respectively. Short-term (5 year) survival after ASA (SMR=0.61, p=0.48) was comparable to that of the general population. CONCLUSION: Long-term follow-up of septal reduction strategies in obstructive HCM reveals that surgical myectomy and ASA are effective for symptom relief and LVOT gradient reduction and are associated with favorable survival. While overall prognosis for the community HCM population is similar to the general population, the need for surgical myectomy may identify a sub-group with poorer long-term prognosis. We await long-term outcomes of more extensive myectomy approaches adopted in the past 10 years at major institutions.

Impaired cardiac anti-oxidant activity in diabetes: human and correlative experimental studies.

Increased reactive oxygen species (ROS) are traditionally viewed as arising from the metabolic flux of diabetes, although reduction in the activity of anti-oxidant systems has also been implicated. Among the latter is the major thiol reducing thioredoxin system, the activity of which may be diminished by high glucose-induced expression of its endogenous inhibitor, thioredoxin interacting protein (TxnIP). We assessed TxnIP mRNA/protein expression along with thioredoxin activity in human right atrial biopsy specimens from subjects with and without diabetes undergoing coronary artery grafting. In correlative experimental studies, we examined TxnIP expression in both type 1 and type 2 rodent models of diabetic cardiomyopathy. Finally, we used in vitro gene silencing to determine the contribution of changes in TxnIP abundance to the high glucose-induced reduction in thioredoxin activity. In human right atrial biopsies, diabetes was associated with a >30-fold increase in TxnIP gene expression and a 17 % increase in TxnIP protein expression (both p < 0.05). This was associated with a 21 % reduction in thioredoxin activity when compared to human non-diabetic cardiac biopsy samples (all p < 0.05). In correlative animal studies, both type 1 and type 2 diabetic rats demonstrated a significant increase in TxnIP mRNA and reduction in thioredoxin activity when compared to non-diabetic animals (all p < 0.05). This was associated with a significant increase in ROS (p < 0.05 when compared with control). In cultured cardiac myocytes, high glucose increased ROS and TxnIP mRNA expression, in association with a reduction in thioredoxin activity (p < 0.01). These findings were abrogated by TxnIP small interfering RNA (siRNA). Scrambled siRNA had no effect upon ROS or TxnIP expression. High glucose reduces thioredoxin activity and increases ROS via TxnIP overexpression. These findings suggest that impaired thiol reductive capacity, through altered TxnIP expression, contributes to increased ROS in the diabetic heart.

MdoR is a novel positive transcriptional regulator for the oxidation of methanol in Mycobacterium sp. strain JC1.

Mycobacterium sp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for methanol oxidation. The second open reading frame (mdoR) upstream of, and running divergently from, the mdo gene was identified as a gene for a TetR family transcriptional regulator. The N-terminal region of MdoR contained a helix-turn-helix DNA-binding motif. An electrophoretic mobility shift assay (EMSA) indicated that MdoR could bind to a mdo promoter region containing an inverted repeat. The mdoR deletion mutant did not grow on methanol, but growth on methanol was restored by a plasmid containing an intact mdoR gene. In DNase I footprinting and EMSA experiments, MdoR bound to two inverted repeats in the putative mdoR promoter region. Reverse transcription-PCR indicated that the mdoR gene was transcribed only in cells growing on methanol, whereas ß-galactosidase assays showed that the mdoR promoter was activated in the presence of methanol. These results indicate that MdoR serves as a transcriptional activator for the expression of mdo and its own gene. Also, MdoR is the first discovered member of the TetR family of transcriptional regulators to be involved in the regulation of the methanol oxidation, as well as to function as a positive autoregulator.

The cardiac (pro)renin receptor is primarily expressed in myocyte transverse tubules and is increased in experimental diabetic cardiomyopathy.

BACKGROUND: The pro(renin) receptor is a 350 amino acid transmembrane protein, that on ligand binding, increases the catalytic efficiency of angiotensinogen cleavage by both prorenin and renin, augmenting angiotensin I formation at the cell surface. While implicated in a broad range of diseases, studies to date have focused on the kidney, particularly in the diabetic context. We sought to examine the site-specific expression of the pro(renin) receptor within the heart. METHODS: Using confocal microscopy, site-specific markers and transmission electron microscopy we assessed the location of the pro(renin) receptor in the heart at both cellular/sub-cellular levels. We assessed pro(renin) receptor expression in the setting of disease and blockade of the renin-angiotensin system, using the TGR[m(Ren2)-27] model of diabetic cardiomyopathy and the direct renin inhibitor, aliskiren. RESULTS: The pro(renin) receptor was found predominantly at the Z-disc and dyad of cardiac myocytes coinciding closely with the distributions of the vacuolar H⁺-ATPase and ryanodine receptor, known to be located within T-tubules and the sarcoplasmic reticulum's terminal cisternae, respectively. Pro(renin) receptor mRNA/protein abundance were increased ∼3-fold in the hearts of diabetic rats in association with diastolic dysfunction, myocyte hypertrophy and interstitial fibrosis (all P < 0.01). Direct renin inhibition reduced cardiac pro(renin) receptor expression in association with improved cardiac structure/function (all P < 0.05). CONCLUSION: Together, these findings are consistent with the notion that the pro(renin) receptor is a component of the vacuolar H⁺-ATPase, and that like the latter, is increased in the setting of cardiac stress and lowered by the administration of an ostensibly cardioprotective agent.

Terrabacter carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete.

A bacterial strain, PY2(T), capable of oxidizing carbon monoxide, was isolated from a soil sample collected from a roadside at Yonsei University, Seoul, Korea. On the basis of 16S rRNA gene sequence analysis, strain PY2(T) was shown to belong to the genus Terrabacter and was most closely related to Terrabacter lapilli LR-26(T) (99.1 % similarity). Strain PY2(T) was characterized chemotaxonomically as having iso-C(15 : 0) as the predominant fatty acid, MK-8(H(4)) as the major menaquinone, ll-diaminopimelic acid as the diagnostic diamino acid of the cell wall, as possessing a polar lipid profile that included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and unknown amino-containing phosphoglycolipids, and having a DNA G+C content of 75.6 mol%. DNA-DNA relatedness values between strain PY2(T) and the type strains of T. lapilli, Terrabacter tumescens, Terrabacter terrae and Terrabacter aerolatus were 20.0 %, 22.9 %, 35.9 % and 64.5 %, respectively. Based on the combined evidence from the phylogenetic analyses, chemotaxonomic data and DNA-DNA hybridization experiments, it is proposed that strain PY2(T) represents a novel species for which the name Terrabacter carboxydivorans sp. nov. is proposed. The type strain is PY2(T) (=KCCM 42922(T)=JCM 16259(T)).

Identification of trans- and cis-control elements involved in regulation of the carbon monoxide dehydrogenase genes in Mycobacterium sp. strain JC1 DSM 3803.

The cutR gene was identified 314 bp upstream of the divergently oriented cutB1C1A1 operon encoding carbon monoxide (CO) dehydrogenase in Mycobacterium sp. strain JC1. Its deduced product was composed of 320 amino acid residues with a calculated molecular mass of 34.1 kDa and exhibits a basal sequence similarity to the regulatory proteins belonging to the LysR family. Using a cutR deletion mutant, it was demonstrated that CutR is required for the efficient utilization of CO by Mycobacterium sp. strain JC1 growing with CO as the sole source of carbon and energy. CutR served as a transcriptional activator for expression of the duplicated cutBCA operons (cutB1C1A1 and cutB2C2A2) and was involved in the induction of the cutBCA operons by CO. The cutBCA operons were also subjected to catabolite repression. An inverted repeat sequence (TGTGA-N(6)-TCACA) with a perfect match with the binding motif of cyclic AMP receptor protein was identified immediately upstream of and overlapping with the translational start codons of cutB1 and cutB2. This palindrome sequence was shown to be involved in catabolite repression of the cutBCA operons. The transcription start point of cutR was determined to be the nucleotide G located 36 bp upstream of the start codon of cutR. Expression of cutR was higher in Mycobacterium sp. strain JC1 grown with glucose than that grown with CO.

Identification and functional characterization of a gene for the methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase from Mycobacterium sp. strain JC1 (DSM 3803).

Mycobacterium sp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for primary methanol oxidation. Purified MDO oxidizes ethanol and formaldehyde as well as methanol. The Mycobacterium sp. strain JC1 gene for MDO (mdo) was cloned, sequenced, and determined to have an open reading frame of 1272 bp. Northern blot and promoter analysis revealed that mdo transcription was induced in cells grown in the presence of methanol. Northern blotting together with RT-PCR also showed that the mdo gene was transcribed as monocistronic mRNA. Primer extension analysis revealed that the transcriptional start site of the mdo gene is located 21 bp upstream of the mdo start codon. An mdo-deficient mutant of Mycobacterium sp. strain JC1 did not grow with methanol as a sole source of carbon and energy.

Cloning and expression analysis of the duplicated genes for carbon monoxide dehydrogenase of Mycobacterium sp. strain JC1 DSM 3803.

Carbon monoxide dehydrogenase (CO-DH) is an enzyme catalysing the oxidation of CO to carbon dioxide in Mycobacterium sp. strain JC1 DSM 3803. Cloning of the genes encoding CO-DH from the bacterium and sequencing of overlapping clones revealed the presence of duplicated sets of genes for three subunits of the enzyme, cutB1C1A1 and cutB2C2A2, in operons, and a cluster of genes encoding proteins that may be involved in CO metabolism, including a possible transcriptional regulator. Phylogenetic analysis based on the amino acid sequences of large subunits of CO-DH suggested that the CO-DHs of Mycobacterium sp. JC1 and other mycobacteria are distinct from those of other types of bacteria. The growth phenotype of mutant strains lacking cutA genes and of a corresponding complemented strain showed that both of the duplicated sets of CO-DH genes were functional in this bacterium. Transcriptional fusions of the cutB genes with lacZ revealed that the cutBCA operons were expressed regardless of the presence of CO and were further inducible by CO. Primer extension analysis indicated two promoters, one expressed in the absence of CO and the other induced in the presence of CO. This is believed to be the first report to show the presence of multiple copies of CO-DH genes with identical sequences and in close proximity in carboxydobacteria, and to present the genetic evidence for the function of the genes in mycobacteria.

Tsukamurella carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete.

A Gram-positive, slightly acid-alcohol-fast, carbon monoxide-oxidizing bacterium, strain Y2(T), was isolated from a soil sample collected from a roadside in Seoul, Korea. On the basis of 16S rRNA gene sequence comparative analyses, strain Y2(T) was shown to belong to the genus Tsukamurella and was most closely related to Tsukamurella tyrosinosolvens DSM 44234(T) (GenBank accession no. AY238514; 99.8 %). The predominant fatty acids were C(18 : 1)omega9c and C(16 : 0). The cell-wall peptidoglycan of strain Y2(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. Strain Y2(T) contained galactose and arabinose as the whole cell sugars. The DNA G+C content was 77 mol%. The DNA-DNA relatedness value between strain Y2(T) and T. tyrosinosolvens DSM 44234(T) was 62.7 %. Based on the combination of the carbon source utilization pattern, fatty acid profile, cell-wall chemotype, DNA G+C content and DNA-DNA hybridization experiments, it is proposed that strain Y2(T) (=KCCM 42885(T)=JCM 15482(T)) represents the type strain of a novel species, Tsukamurella carboxydivorans sp. nov.

Pseudonocardia carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete, and an emended description of the genus Pseudonocardia.

A bacterial strain, Y8(T), capable of oxidizing carbon monoxide, was isolated from a soil sample collected from a roadside in Seoul, Korea. On the basis of 16S rRNA gene sequence similarity analyses, strain Y8(T) was shown to belong to the genus Pseudonocardia and was related most closely to the type strain of Pseudonocardia alni (99.6 % similarity). The cells were aerobic and stained Gram-positive, with white aerial mycelium and brown substrate mycelium. The predominant fatty acids were 16 : 0 iso and 16 : 1 iso. The cell-wall peptidoglycan of strain Y8(T) contained meso-diaminopimelic acid. The DNA G+C content was 77 mol%. Strain Y8(T) contained MK-9 as the major menaquinone, which is different from the major menaquinone reported previously in the genus Pseudonocardia, MK-8(H(4)). DNA-DNA relatedness between strain Y8(T) and the type strains of P. alni and Pseudonocardia antarctica was respectively 10 and 63 %. Based on phylogenetic, morphological and chemotaxonomic evidence, it is proposed that strain Y8(T) (=KCCM 42678(T) =JCM 14827(T)) be classified as the type strain of a novel species, Pseudonocardia carboxydivorans sp. nov. An emended description of the genus Pseudonocardia is also presented.

Association of atrial nicotinamide adenine dinucleotide phosphate oxidase activity with the development of atrial fibrillation after cardiac surgery.

OBJECTIVES: Our goal was to evaluate the role of myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and plasma markers of oxidative stress in the pathogenesis of post-operative atrial fibrillation (AF). BACKGROUND: Atrial fibrillation is a common complication of cardiac surgery, leading to increased morbidity and prolonged hospitalization. Experimental evidence suggests that oxidative stress may be involved in the pathogenesis of AF; however, the relevance of this putative mechanism in patients undergoing cardiac surgery is unclear. METHODS: We measured basal and NADPH-stimulated superoxide production in right atrial appendage samples from 170 consecutive patients undergoing conventional coronary artery bypass surgery. Plasma markers of lipid and protein oxidation (thiorbabituric acid-reactive substances, 8-isoprostane, and protein carbonyls) were also measured in blood samples drawn from a central line before surgery and after reperfusion. RESULTS: Patients who developed AF after surgery (42%) were older and had a significantly increased atrial NADPH oxidase activity than patients who remained in sinus rhythm (SR) (in relative light units/s/mug protein: 4.78 +/- 1.44 vs. 3.53 +/- 1.04 in SR patients, p < 0.0001). Plasma markers of lipid and protein oxidation increased significantly after reperfusion; however, neither pre-operative nor post-operative measurements differed between patients who developed AF and those who remained in SR after surgery. Multivariate analysis identified atrial NADPH oxidase activity as the strongest independent predictor of post-operative AF (odds ratio 2.41; 95% confidence interval 1.71 to 3.40, p < 0.0001). CONCLUSIONS: Atrial NADPH oxidase activity is independently associated with an increased risk of post-operative AF, suggesting that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after cardiac surgery.

Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803.

Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78,197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of approximately 78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and beta-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.

Selective adsorption of phenanthrene dissolved in surfactant solution using activated carbon.

Selective adsorption of a hazardous hydrophobic organic compound (HOC) by activated carbon as a means of recovering surfactants after a soil washing process was investigated. As a model system, phenanthrene was selected as a representative HOC and Triton X-100 as a nonionic surfactant. Three activated carbons that differed in size (Darco 20-40 (D20), 12-20 (D12) and 4-12 (D4) mesh sizes) were used in adsorption experiments. Adsorption of surfactant onto activated carbon showed a constant maximum above the critical micelle concentration, which were 0.30, 0.23, 0.15 g g(-1) for D20, D12, and D4, respectively. Selectivity for phenanthrene to Triton X-100 was much higher than 1 over a wide range of activated carbon doses (0-6 g l(-1)) and initial phenanthrene concentrations (10-110 mg l(-1)). Selectivity generally increased with decreasing particle size, increasing activated carbon dose, and decreasing initial concentration of phenanthrene. The highest selectivity was 74.9, 57.3, and 38.3 for D20, D12, and D4, respectively, at the initial conditions of 10 mg l(-1) phenanthrene, 5 g l(-1) Triton X-100 and 1g l(-1) activated carbon. In the case of D20 at the same conditions, 86.5% of the initial phenanthrene was removed by sorption and 93.6% of the initial Triton X-100 remained in the solution following the selective adsorption process. The results suggest that the selective adsorption by activated carbon is a good alternative for surfactant recovery in a soil washing process.

Angiotensin-converting enzyme genotype predicts cardiac and autonomic responses to prolonged exercise.

OBJECTIVES: The purpose of this study was to investigate the phenomenon of left ventricular (LV) dysfunction after ultraendurance exercise. BACKGROUND: Subclinical LV dysfunction in response to endurance exercise up to 24 h duration has been described, but its mechanism remains elusive. METHODS: We tested 86 athletes before and after the Adrenalin Rush Adventure Race using echocardiography, impedance cardiography, and plasma immunoassay. RESULTS: At baseline, athletes demonstrated physiology characteristic of extreme endurance training. After 90 to 120 h of almost-continuous exercise, LV systolic and diastolic function declined (fractional shortening before the race, 39.6 +/- 0.65%; after, 32.2 +/- 0.84%, p < 0.001; mitral inflow E-wave deceleration time before the race, 133 +/- 5 ms; after, 160 +/- 5 ms, n = 48, p < 0.001) without change in loading conditions as defined by LV end-diastolic dimension and total peripheral resistance estimated by thoracic impedance. There was a compensatory increase in heart rate (before, 55 +/- 1.3 beats/min; after, 59 +/- 1.5 beats/min, p = 0.05), which left cardiac output unchanged, as well as significant-but-subclinical increases in brain natriuretic peptide and troponin I. In addition, we found that athletes who were homozygous for the intron-16 insertion polymorphism of the angiotensin-converting enzyme (ACE) gene exhibited a significantly greater decrease in fractional shortening than athletes who were homozygous for the deletion allele. Heterozygotes showed an intermediate phenotype. In addition, the deletion group manifest an enhanced sympathovagal balance after the race, as evidenced by greater power in the low-frequency component of blood pressure variability. CONCLUSIONS: The ACE genotype predicts the extent of reversible subclinical LV dysfunction after prolonged exercise and is associated with a differential postactivity augmentation of sympathetic nervous system function that may explain it.