An increase in high-density lipoprotein cholesterol after weight loss intervention is associated with long-term maintenance of reduced visceral abdominal fat.

OBJECTIVES: It is generally agreed that excess abdominal fat, in particular visceral abdominal fat (VAF), is related to an increased risk for obesity-related complications. We examined the association between metabolic risk factors and maintaining VAF after weight loss intervention. METHODS: A total of 54 postmenopausal, obese women who achieved a VAF loss of at least 10% from their baseline values during a 14-week intervention were enrolled as subjects. Body weight, VAF assessed by CT scans, and metabolic risk factors (that is, blood pressure, lipids and glucose) were measured at baseline (week 0), post-intervention (week 15), and at a 2-year follow-up (week 105). The subjects were divided into two groups according to their changes in VAF between weeks 15 and 105 (follow-up period): (1) VAF gainers (VAF changes > 0 cm², n=28) or (2) VAF maintainers (VAF changes ≤ 0 cm², n=26). RESULTS: The mean change in VAF of all subjects during the 14-week intervention was -34±16 cm² (-29.7±12.3%) (P<0.01). Along with this change, improvements (P<0.05) were observed in all metabolic risk factors except for high-density lipoprotein cholesterol (HDLC). During the follow-up period, there were interactions between the two VAF groups in HDLC, triglycerides (TG) and total cholesterol (TC)/HDLC ratio (all P<0.01). In particular, the HDLC of VAF maintainers improved, and the value at week 105 exceeded baseline level (P<0.01). However, systolic and diastolic blood pressure, TC and low-density lipoprotein cholesterol in the VAF maintainers increased (all P<0.05) back to their mean baseline level despite a further decrease in their VAF during the follow-up period (P<0.01). CONCLUSIONS: This study shows that long-term maintenance of VAF after weight loss intervention is associated with improvements in HDLC and TG among obese, postmenopausal women.

Early-stage dynamics of light-matter interaction leading to the insulator-to-metal transition in a charge ordered organic crystal.

Ultrafast dynamics of the light-matter interaction in a charge-ordered molecular insulator α-(BEDT-TTF)2I3 were studied by pump-probe spectroscopy using few-optical-cycle infrared pulses (pulse width 12 fs). Coherent oscillation of the correlated electrons and subsequent Fano destructive interference with intramolecular vibration were observed in time domain; the results indicated a crucial role for electron-electron interplay in the light-matter interaction leading to the photoinduced insulator-to-metal transition. The qualitative features of this correlated electron motion were reproduced by calculations based on exact many-electron-phonon wave functions.

Human C4b-binding protein, C4bp. Chymotryptic cleavage and location of the 48 kDa active fragment within C4bp.

C4bp, a regulator of the classical pathway of complement system, is composed of 6-8 disulfide-linked subunit chains of 75 kDa. Upon incubation with chymotrypsin, C4bp was rapidly cleaved into a nicked C4bp, composed of disulfide-linked 48 kDa and 27 kDa fragments. Subsequent slow cleavage on the 27 kDa fragment resulted in the liberation of the active site-containing 48 kDa fragment from the nicked C4bp. The N-terminal amino acid sequence of the 48 kDa fragment was identical to that of the parent subunit chain of C4bp, indicating that the 48 kDa active fragment was released from the N-terminal side of the parent subunit chain. Based on these results, a possible gross structure of C4bp is proposed.

Functional analysis of antibacterial activity of Bacillus amyloliquefaciens phage endolysin against Gram-negative bacteria.

To analyze the antibacterial activity of Bacillus amyloliquefaciens phage endolysin, nine deletion derivatives of the endolysin were constructed. Each deletion mutant was overexpressed, purified and characterized. The catalytic domain was located on the N-terminal region and the C-terminus had an affinity with the bacterial envelope. The enzymatic activity remained in spite of the deletion of the C-terminal 116-amino acid region; however, the antibacterial activity was lost. These results indicate that antibacterial action requires both the C-terminal cell-binding and the N-terminal enzymatic activities.

Evidence for a novel Chlorella virus-encoded alginate lyase.

To clone the genes encoding lysis protein from a Chlorella virus, water samples were collected from 13 aquatic environments located in the Kanto area of Japan. Eight water samples contained plaque-forming viruses on Chlorella sp. NC64A, but no virus was detected in the other five samples. A novel Chlorella virus, CVN1, was isolated from the Inba-numa marsh sample. CVN1 genomic DNA was partially digested and shotgun cloned into pUC118 to identify the genomic region responsible for the lytic phenotype on Chlorella sp. NC64A. A DNA fragment which encoded two ORFs, ORF1 and ORF2, was obtained by antialgal assay. The ORF2 gene product, CL2, consisted of 333 amino acids showing antialgal activity not only on the original host of Chlorella sp. NC64A, but also on the heterogeneous hosts of Chlorella vulgaris C-27 and C. vulgaris C-207. CL2 showed a weak homology (19.8% amino acid identity) to mannuronate lyase SP2 from Turbo cornutus. CL2 in Escherichia coli cells was purified using a nickel chelate column. Lyase activity of purified CL2 on alginic acid was observed in an enzyme assay. The specific activity of purified CL2 was 2.1x10(-2) U mg(-1), the optimum pH for enzymatic activity was 10.5, and Ca(2+) was required for enzyme activity. This is the first report of a Chlorella virus protein with lyase activity.

Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes.

Self-disruptive Escherichia coli that produces foreign target protein was developed. E. coli was co-transformed with two vector plasmids, a target gene expression vector and a lysis gene expression vector. The lytic protein was produced after the expression of the target gene, resulting in simplification of the cell disruption process. In this study, the expression of cloned T4 phage gene e or t was used for the disruption of E. coli that produced beta-glucuronidase (GUS) as a model target protein. The expression of gene e did not lead to prompt cell disruption but weakened the cell wall. Resuspension with deionized water facilitated cell lysis, and GUS activity was observed in the resuspended liquid. Expression of gene e at mid logarithmic growth phase was the optimal induction period for GUS production and release. On the other hand, the expression of gene t induced immediate cell lysis, and intracellular GUS was released to the culture medium. Maximum GUS production was obtained when gene t was induced at late logarithmic growth phase.

Characterization of oxygen-dependent lysis of Escherichia coli cells infected by bacteriophage T4.

Infection of Escherichia coli cells by T4 phage at a multiplicity of infection (MOI) of 0.01 caused inhibition of cell lysis for up to 4 h. Such cells grown under aerobic condition were lysed by external stimuli such as cold shock, osmotic shock or addition of toxic substances, e.g., carbonyl cyanide m-chlorophenylhydrazone (CCCP). However, the effects of these external stimuli were reduced by transferring the cells to static incubation, by which dissolved oxygen was consumed by the cells within 10 min. The cells became insensitive to such external stimuli when the culture was deoxygenated with nitrogen gas. Following infection with a lysozyme amber mutant, eL1a, the cell membrane permeability was found to be increased either by cold shock or osmotic shock treatment of cells grown under aerobic conditions, but not in cells transferred to the static incubation. Oxygen limitation was suggested to enhance membrane stability in relation to cell lysis following the cold or osmotic shock treatment.

Cloning and expression of a gene encoding the lytic functions of Bacillus amyloliquefaciens phage: Evidence of an auxiliary lysis system.

A bacteriophage specific to Bacillus amyloliquefaciens, a gram-positive bacterium, was isolated from a local sewage treatment center. Using a lysis assay, a gene, lys1521, was isolated and its nucleotide sequence revealed one open reading frame of 375 bp. Homology studies showed amino acid alignment similarity with gene 5A of Bacillus subtilis phages PZA and phi29. Overexpression of the cloned gene yielded a 13 kDa protein corresponding to the predicted gene product. Despite the fact that no significant homology with known cell wall lytic enzymes was apparent, the lytic profile obtained in an in vivo expression assay showed that lys1521 had cell wall hydrolysis activity. This is a significant revelation since the function of the homologous gene 5A product of phage phi29 has been suggested to be required for the in vivo elongation of phage DNA replication. The lys1521 gene could be evidence of the presence in gram-positive bacteriophages of a third lysis gene in addition to the well characterized two-step lysis system.

Stimulating accumulation of nitrifying bacteria in porous carrier by addition of inorganic carbon in a continuous-flow fluidized bed wastewater treatment reactor.

Porous polyurethane carrier particles have been successfully applied for microbial immobilization to simultaneously remove carbonaceous and nitrogenous substances from wastewater by a fill-and-draw operation. This reactor system was extended to a continuous-flow operation mode, by which inorganic carbon (IC) was supplemented in order to stimulate the growth of autotrophic nitrifying bacteria. By addition of sodium bicarbonate, the ammonia oxidation reaction proceeded remarkably in the porous particle fluidized bed reactor, while a small increase in the nitrification was observed in a reactor with suspended microbes. Dissolved oxygen profile was obtained using an oxygen microelectrode to measure the microbial consumption of oxygen in the porous carrier. The size of ammonia-oxidizing bacterial populations in the carrier was proportional to the volume of the aerobic region of the carrier. The aerobic region decreased with the increase in sodium bicarbonate concentration, which improved the ammonia-oxidizing activity of retained nitrifiers in the carrier. The maximum ammonia oxidation rate was up to 55.6 gN/m3/h within the aerobic region of the carrier under the following feed conditions: 100 mg/l of total organic compound, 55 mg/l of ammonium concentration and 48 mg/l of inorganic carbon.

Enhanced microbial adaptation to p-nitrophenol using activated sludge retained in porous carrier particles and simultaneous removal of nitrite released from degradation of p-nitrophenol.

In order to examine the microbial degradation of p-nitrophenol (PNP) by a mixed culture system and simultaneous removal of nitrite released via the degradation, an activated sludge retained in porous carrier particles and a suspension culture as a control were acclimated to artificial sewage containing PNP as the sole carbon source. The adaptation of microbes retained in porous carrier particles to PNP was faster than that of suspended microbes by more than 20 d. After microbial adaptation to PNP, it was degraded completely without significant accumulation of intermediate metabolites. The PNP degradation activity of the retained microbes was more than 2 times higher than that of the suspended microbes. By increasing the retained microbial concentration, nitrite released from the degraded PNP was removed by denitrification. This research demonstrates that using microbes retained in porous carrier particles is not only effective for reduction of acclimation time but also enables simultaneous removal of the nitrogen compounds resulting from the degradation of nitroaromatics.

Structural analysis of a biofilm which enhances carbon steel corrosion in nutritionally poor aquatic environments.

Carbon steel coupons were exposed to nutritionally-poor synthetic wastewater inoculated with activated sludge from a municipal waste water plant. Biofilm formation was observed after one day incubation, and the thickness of the film increased proportionally with the incubation period. Mass loss of the coupons was also proportional to the incubation time, and reached 70.4 (mg/cm2) after incubation for 140 d. The observed mass loss was 5 times as much as that under sterile conditions. To characterize the microbiologically influenced corrosion (MIC) of carbon steel, structural analysis of the biofilm was performed. Rapid decrease in the dissolved oxygen (DO) concentration in the zone near the surface of the biofilm was observed by a microelectrode mounted on a micromanipulator. Heterogeneous distribution of the DO concentration on the surface of the steel plate was observed after multiple analyses. The heterogeneous structure of the biofilm composed of viable cells, inanimate objects, voids and pores was elucidated by confocal scanning laser microscopy. Concentrations of both aerobic bacteria and sulphur-reducing bacteria in the biofilm decreased with the incubation time, indicating that the increase in the biofilm thickness reflected an increase in the density of dead microbial cells or in extracellular polymer accumulation by the microbes. The average roughness of the metal surface observed after 112 d of incubation was +/-7.14 microm, which was 14.1% of the average thickness of the coupons. These observations indicated that uneven distribution of the DO profile and the cell concentration were critical for MIC of the carbon steel.

Aggregate formation of rCHO cells and its maintenance in repeated batch culture in the absence of cell adhesion materials.

Aggregate formation of recombinant Chinese hamster ovary (rCHO) cells capable of producing granulocyte colony-stimulating factor (G-CSF), using medium lacking cell adhesion materials in a repeated batch culture, was examined together with cell growth, cell viability and G-CSF production. The rCHO culture was conducted in a rotary shaker and the medium was changed every five days. The formation of stable cell aggregates with high reproducibility was observed after the first medium change. The size of the cell aggregates (consisting of several 10s to 40,000 cells) formed during the repeated batch culture ranged from 30 to 600 microm. The cell density of the aggregates reached as high as 2 x 10(6) cells/ml and the viability was maintained at more than 80% for 19 d. Changing the medium to avoid glucose exhaustion effectively maintained the cell density, cell viability and G-CSF productivity at high levels.

Antibacterial activity of Bacillus amyloliquefaciens phage endolysin without holin conjugation.

To characterize the enzymatic activity and antibacterial activity of endolysin encoded by a Bacillus amyloliquefaciens phage, the open reading frame encoding endolysin was amplified by PCR and cloned into the expression plasmid pET21d(+). The resultant plasmid was used to transform Escherichia coli JM109(DE3). Production of endolysin in the cytosol facilitated cell lysis without coproduction of holin, which is considered to degrade or alter the cytoplasmic membrane. The phage endolysin was overexpressed and purified. Although the specific activity of the purified phage endolysin towards lyophilized Micrococcus luteus cells was 1/11 of the activity of chicken egg white lysozymes, the endolysin showed stronger antibacterial activity towards E. coli W3110, E. coli JM109(DE3) and Pseudomonas aeruginosa PAO1 than chicken egg white lysozymes. The antibacterial activity of the endolysin towards these three bacterial strains was marked when EDTA was added to the endolysin solution.

Implantation of a mechanical valve within the orifice of a mitral bioprosthesis in a case with severely calcified left atrium--a case report.

The case of a 61-year-old woman with deterioration of mitral bioprosthesis and severe left atrial calcification is presented. Although the implantation of a mechanical valve within the orifice of a mitral bioprosthesis was tried, we found a major pitfall in this method was exact orifice matching.

[Analysis of the cases in which Schizophyllum commune was isolated].

Infections caused by Schizophyllum commune have recently been on the increase. We analyzed cases in which this fungus was isolated from clinical samples from 1991 to 1995. Regular morphological methods were first employed in the identification of S. commune, and when necessary, mating was made with a known S. commune isolate. IgG antibodies against S. commune in the patients' serum were determined by the ELISA method. Profiles of the cases were analyzed and the English-language medical literature was reviewed. S. commune was isolated from 12 patients. Eighty three percent of the patients were female, most of which presented with allergy-related pulmonary diseases, i. e. allergic bronchopulmonary mycosis or mucoid impaction of bronchi. IgG antibodies were detectable in nine of eleven patients examined (82%). Fifty eight percent of the isolates were monokaryotic, and therefore, unidentifiable by regular morphological methods. In contrast, analysis of the English literature disclosed that sinusitis is the most common form of infection, and that hyphal invasion into the tissue was seen in 50% of reported cases. Our study shows that S. commune should be regarded as a pathogenic fungus, and is infecting humans much more frequently than previously assumed. Further investigation is warranted.

Fate of coliphage in waste water treatment process and detection of phages carrying the Shiga toxin type 2 gene.

Abundances of phages specific to Escherichia coli in the wastewater treatment process were analyzed. Relatively abundant coliphages were detected in sewage influent. Phages in the influent were found both suspended in liquid phase and attached on the solid particles. Phage concentration was not reduced in the settling tank without chemical agglutination. Anaerobic followed by aerobic treatment of the sewage reduced concentration of suspended phages. Almost no phage was detected as a suspended form in the aerobic tank. Most of the phages were detected as attaching form and were excluded by aggregation with sludge. Using an experimental approach based on the detection of Shiga toxin 2 (Stx 2) gene by a phage enrichment culture followed by nested PCR, bacteriophages carrying Stx 2 gene were detected in the influent, settling tank, and anaerobic tank. It was revealed that the presence of phages carrying Stx 2 gene is common in sewage and these phages are effectively eliminated through sewage treatment process.

[The treatment of pulmonary aspergilloma with itraconazole].

We evaluated the efficacy of Itraconazole (ITZ), a newly developed triazole antifungal agent, in 5 cases of pulmonary aspergilloma. A daily dose of 100-200 mg of ITZ was given orally for 5 to 20 months. In one patient, administration of ITZ was discontinued because of mild and transient hepatic injury. On computerized tomography and conventional roentgenogram of the chest, improvement was clearly observed in 2 of the remaining 4 cases. Symptomatic improvement, mainly disappearance of airway bleeding, was seen in 2 cases. Thus the overall improvement rate was 50% (two out of 4). No adverse response other than the hepatic injury mentioned above occurred. In one patient, who had two fungus balls previously, one of the fungus balls disappeared completely and the other decreased significantly in size during the course of treatment. In this case, the daily dose of ITZ was larger (200 mg once a day) and the plasma concentration of ITZ was extremely higher (2068 ng/ml) than in other cases (137-330 ng/ml). We conclude that ITZ is an efficacious drug in the treatment of pulmonary aspergilloma, and that this drug may be even more efficacious if we keep higher plasma concentration of ITZ.

[Strategies for preventing stroke after coronary artery bypass grafting].

UNLABELLED: To evaluate the usefulness of our strategy for preventing stroke after CABG, 343 consecutive patients were investigated retrospectively. Patient ages ranged from 32 to 31 years (mean; 63 +/- 9 years). There were 254 males and 59 females. Number of grafts per patient was 1 to 5 (mean 2.4 +/- 0.9 grafts). In 193 patients, internal carotid arteries (ICAs) were preoperatively evaluated by duplex scanning or cerebral angiogram. The degree of atherosclerosis in the ascending aorta was preoperatively examined by plain computed tomography in 181 patients, during surgery by ultrasonography in 75 patients and palpation in all patients. RESULTS: 1. On preoperative examination, there were 26 patients (15.1%) with ICA stenosis greater than 50% and 15 patients (7.8%) with stenosis greater than 75%. Six patients had bilateral ICA stenosis or occlusion greater than 75%. In 26 patients with ICA stenosis greater than 50%, history of stroke was significantly more prevalent than that in 167 patients without ICA stenosis (12 patients: 46.2% vs 22 patients: 13.1%, p < 0.001). In patients with ICA stenosis greater than 75%, 6 patients were symptomatic and 8 were asymptomatic. For these patients, concomitant carotid endarterectomy and CABG were performed in 5, two stage procedures in 7 reconstruction of cerebral perfusion followed by CABG;4, followed by CEA: 3), and CABG alone in 3. There was no stroke in any of these patients. 2. Atherosclerosis of the ascending aorta was found in 69 of 343 patients (20.1%). In these patients, single clamp technique was applied in 50 patients, aortic no touch technique in 12 and CABG without cardiopulmonary bypass in one. The arterial cannulation site was changed to femoral artery in 15 and to axillary artery in 6 patients. Statistical analysis indicated that age (older than 60 years) and history of stroke were significant risk factors for atherosclerotic ascending aorta. 3. There were 3 patients (0.9%) with perioperative stroke caused by embolism from the ascending aorta in one and hypoperfusion of the brain during cardiopulmonary bypass in two. CONCLUSION: Proper treatment of atherosclerotic ascending aorta and carotid occlusion may reduce the incidence of stroke in CABG patients.

Bilateral internal thoracic artery T grafting for coronary artery revascularization. Angiographic assessment and mid-term outcome.

OBJECTIVES: We studied the early outcome of bilateral internal thoracic artery T grafting. METHODS: Coronary artery bypass grafting was studied retrospectively using bilateral internal thoracic artery T grafting in 51 patients. The T graft was made by anastomosing the free right internal thoracic artery to the in-situ left internal thoracic artery. Average patient age was 63.5 +/- 9.9 years, and the average number of anastomoses per patient was 3.6 +/- 0.9. In 35 patients, the right gastroepiploic artery (21 anastomoses in 20 patients), radial artery (1 anastomosis), free left internal thoracic artery (1 anastomosis) and saphenous vein graft (14 anastomoses in 13 patients) were used as additional bypass conduits. RESULTS: Hospital mortality was 0%. The morbidity of stroke was 1.9% (1 patient) and deep sternal infection 0%. Patency of the in-situ left internal thoracic artery was 49/50 anastomoses (98%) and that of the free right internal thoracic artery 81/84 anastomoses (96.4%). Mid-term coronary angiography in 7 patients demonstrated patent anastomosis of the T graft. Acute myocardial infarction unrelated to graft failure occurred in 2 patients during follow-up. Other patients were evaluated by exercise stress tests every year and none exhibited myocardial ischemia in the areas of T graft coronary revascularization. Three-year actuarial survival rate was 100% and freedom from cardiac events 96%. CONCLUSIONS: The bilateral internal thoracic artery T graft provides satisfactory early and mid-term outcomes in properly selected patients.

Acute heart failure due to local dehiscence of aortic wall at aortic valvular commissure.

Spontaneous dehiscence of the aortic wall at the aortic commissure is not recognized as one of the usual pathological causes of aortic regurgitation. We describe the case of a 56-year-old man with hypertension, who experienced acutely progressive congestive heart failure due to massive aortic regurgitation. Local layer dehiscence around the commissure was noted with partial detachment of the commissure resulting in the loss of commissural support with secondary rupture of a non-coronary cusp, which led to massive aortic regurgitation.