A potent allele marker related to low bull conception rate in Japanese Black bulls.

Over the years, there has been considerable variation in the bull conception rate (BCR) of Japanese Black cattle; moreover, several Japanese Black bulls with a low BCR of ≤10% have been identified. However, the alleles responsible for the low BCR are not determined yet. Therefore, in this study, we aimed to identify single-nucleotide polymorphisms (SNPs) for predicting low BCR. To this end, the genome of Japanese Black bulls was comprehensively examined by a genome-wide association study with whole-exome sequencing (WES), and the effect of the identified marker regions on BCR was determined. The WES analysis of six sub-fertile bulls with a BCR of ≤10% and 73 normal bulls with a BCR of ≥40% identified a homozygous genotype for low BCR in Bos taurus autosome 5 in the region between 116.2 and 117.9 Mb. The g.116408653G > A SNP in this region had the most significant effect on the BCR (P-value = 1.0 × 10-23), and the GG (55.4 ± 11.2%) and AG (54.4 ± 9.4%) genotypes in the SNP had a higher phenotype than the AA (9.5 ± 6.1%) genotype for the BCR. The mixed model analysis revealed that g.116408653G > A was related to approximately 43% of the total genetic variance. In conclusion, the AA genotype of g.116408653G > A is a useful index for identifying sub-fertile Japanese Black bulls. Some positive and negative effects of SNP on the BCR were presumed to identify the causative mutations, which can help evaluate bull fertility.

Paclitaxel plus Carboplatin Chemotherapy for Primary Peritoneal Carcinoma: A Study of 22 Cases and Comparison with Stage III-IV Ovarian Serous Carcinoma.

The aim of this study was to assess the clinical characteristics and outcome of patients with either primary peritoneal carcinoma (PPC) or ovarian serous carcinoma (OSC) treated with paclitaxel plus carboplatin chemotherapy. We retrospectively identified 22 PPC patients and 55 stage III-IV OSC patients treated between 2002 and 2007. After exploratory laparotomy, all patients received paclitaxel and carboplatin every 3 weeks, with the goal of optimal cytoreduction. There were no statistically significant differences between the PPC and OSC groups with regard to tumor stage, residual tumor after debulking surgery (initial or interval), serum cancer antigen (CA) 125 levels at diagnosis, and completion of first-line chemotherapy. The progression-free survival (PFS) durations were 12.7 months (95% CI, 6.3-18.5) in the patients with PPC and 15.9 months (95% CI, 13.3-18.5) in those with OSC (p = 0.016). However, the median survival durations were 26.5 months (95% CI, 14.6-38.3) in the patients with PPC and 38 months (95% CI, 23.8-53.8) in those with OSC (p = 0.188). Survival was longer for all patients whose CA125 levels normalized to 26 U/ml during and after treatment. Overall survival (OS) of the patients with PPC was similar to that of the patients with OSC, suggesting that management for advanced-stage OSC would be similar to that for PPC. The combination of optimal debulking with paclitaxel plus carboplatin chemotherapy may offer patients the most effective treatment. The CA125 nadir after cytoreductive surgery can be considered a prognostic factor for OS and PFS in patients with PPC.

Bacterial community dynamics during reduction of odorous compounds in aerated pig manure slurry.

AIMS: To study the microbial community responsible for the reduction of the polluting load during aerobic digestion of pig slurry. METHODS AND RESULTS: We analysed bacterial succession by nonculture-based methods and determined the physicochemical parameters and polluting substances during 6 days of aerobic digestion. The bacterial subpopulations evolved by aeration, predominantly Bacillus spp., degraded organic matter and vigorously consumed oxygen, as indicated by low oxidation-reduction potential (ORP). In this phase, the volatile fatty acid (VFA) levels drastically decreased, and VFAs were almost depleted on day 4. Simultaneously, the ammonia concentration decreased to its lowest level on day 4; thereafter, it increased until the end of the process. After the decrease in the total organic carbon content in the supernatant of the decomposed slurry, the ORP increased (approximately 0 mV), and the microbial community showed an abundance of lineages belonging to the phylum Proteobacteria. CONCLUSIONS: Bacillus was the predominant member of the bacterial community driving the VFA-removal process. Their predominance was related to the presence of available carbon, including VFAs and changes in ORP. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the relationships among the involved microbes, polluting materials and physicochemical parameters will aid process design and retrofitting of the process.

A randomized study of screening for ovarian cancer: a multicenter study in Japan.

Ovarian cancer is common in women from developed countries. We designed a prospective randomized controlled trial of ovarian cancer screening to establish an improved strategy for the early detection of cancers. Asymptomatic postmenopausal women were randomly assigned between 1985 and 1999 to either an intervention group (n = 41,688) or a control group (n = 40,799) in a ratio of 1:1, with follow-up of mean 9.2 years, in Shizuoka district, Japan. The original intention was to offer women in the intervention group annual screens by gynecological examination (sequential pelvic ultrasound [US] and serum CA125 test). Women with abnormal US findings and/or raised CA125 values were referred for surgical investigation by a gynecological oncologist. In December 2002, the code was broken and the Shizuoka Cohort Study of Ovarian Cancer Screening and Shizuoka Cancer Registry were searched to determine both malignant and nonmalignant diagnoses. Twenty-seven cancers were detected in the 41,688-screened women. Eight more cancers were diagnosed outside the screening program. Detection rates of ovarian cancer were 0.31 per 1000 at the prevalent screen and 0.38-0.74 per 1000 at subsequent screens; they increased with successive screening rounds. Among the 40,779 control women, 32 women developed ovarian cancer. The proportion of stage I ovarian cancer was higher in the screened group (63%) than in the control group (38%), which did not reach statistical significance (P = 0.2285). This is to our knowledge the first prospective randomized report of the ovarian cancer screening. The rise in the detection of early-stage ovarian cancer in asymptomatic postmenopausal women is not significant, but future decisions on screening policy should be informed by further follow-up from this trial.

Changes in bacterial community during fermentative hydrogen and acid production from organic waste by thermophilic anaerobic microflora.

AIMS: Changes in fermentation pattern during the treatment of organic wastes containing solid materials by thermophilic anaerobic microflora were investigated with respect to product formation and bacterial community structure during hydrogen production. METHODS AND RESULTS: Anaerobic microflora enriched from sludge compost was cultivated using artificial garbage slurry in a continuous flow-stirred tank reactor. Product formation varied depending on pH and hydraulic retention time (HRT) applied. Community analysis by terminal restriction fragment length polymorphism and clone library analysis of polymerase chain reaction-amplified bacterial 16S rDNA indicated that difference in the fermentative product distribution could be caused by different populations of micro-organisms in the microflora. CONCLUSION: Hydrogen fermentation with acetate/butyrate formation was optimized at <1.0 d HRT at pH 5.0 and 6.0. Thermoanaerobacterium thermosaccharolyticum was the dominant hydrogen-producing micro-organism. Conversely, unidentified organisms became dominant after 4.0 d HRT at pH 7.0 and 8.0, where relatively high-solubilization efficiency of solid materials was observed with no production of hydrogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing product formation in the fermentation of solid organic wastes by a mixed population of micro-organisms. Various fermentation patterns including hydrogen fermentation were characterized and evaluated from engineering and microbial aspects.

Activation of mammalian target of rapamycin in postmenopausal ovarian endometriosis.

The purpose of this study was to determine whether Akt and mammalian target of rapamycin (mTOR), downstream targets of phosphatidylinositol 3-kinase, are activated in endometriosis and ovarian cancer specimens. We measured total and phosphorylated levels of Akt and mTOR from 17 frozen ovarian cancers and 15 benign endometriosis specimens (nine from premenopausal women and six from postmenopausal women) by quantitation of signals from western blots using antibodies against these proteins. Elevated phospho-Akt was detected in ovarian cancer versus endometriosis specimens from premenopausal women and endometriosis specimens from postmenopausal women (2.3 +/- 0.45 versus 0.10 +/- 0.06 and 0.17 +/- 0.11; P < 0.05) when the western blot signal of activated kinase was normalized to total kinase levels. Elevated phospho-mTOR was detected in ovarian cancer and postmenopausal endometriosis versus premenopausal endometriosis (0.52 +/- 0.19 and 0.46 +/- 0.29 versus 0.13 +/- 0.08; P < 0.05). Expression of total kinases (normalized to beta-actin) was higher in carcinoma versus endometriosis specimens. Elevation of the active mTOR was specifically detected in postmenopausal endometriosis.

Characteristics of sediments in a newly constructed reservoir in Japan.

The sediment formation mechanisms of a newly constructed reservoir in Ehime, Japan were evaluated by characterizing the soil particles (SP) and particulate phosphorus (PP) in the runoff and reservoir sediments. The SP and PP loads from the runoffs of the main river in the watershed considerably increased, when the specific discharge rates were over 300 l/s/km2 (high flow conditions). When the specific discharge rates exceeded over 300 l/s/km2, 19% of the watershed generated over 80% of the SP and PP loads. When the specific discharge rates were under 300 l/s/km2 (low flow conditions), the contributions of the previously mentioned 19% area to the SP and PP loads were smaller. Significant amounts of smectite were found in the sediments in the reservoir and in the soil samples obtained at the forest exposed area in this 19% area while it was negligible in citrus orchards and paddy fields that constituted the remaining land surfaces. The forest area exposed by recent landslides was significant for the SP and PP in the reservoir. Judging from the outcomes, land use information alone may not be sufficient to detect critical sources of SP and PP in the runoffs and reservoirs. To identify and confirm crucial areas for the SP and PP in the runoffs, the investigations should be conducted under high flow conditions and the composition of clay minerals in the sediments should be checked against the clay mineral distributions of soils in the watershed.

Microbial community changes during organic solid waste treatment analyzed by double gradient-denaturing gradient gel electrophoresis and fluorescence in situ hybridization.

The bacterial community present during semicontinuous treatment of organic solid waste under alkaline and high-temperature conditions was studied. PCR-amplified 16S rDNA fragments were analyzed by double gradient-denaturing gradient gel electrophoresis (DGGE). The band pattern was stable during the steady state of the treatment phase, and the major bands resulting from individual treatments had the same DNA sequence with good reproducibility. No sequence in the DNA database of isolated bacteria showed close similarity to this sequence, the closest relative being Bacillus licheniformis with less than 97% similarity. The conditions for fluorescence in situ hybridization (FISH) were determined without the need to obtain extracts of the bacterial cells. An oligonucleotide probe was designed to detect the microorganisms found in the DGGE analysis. FISH analysis showed that the bacterium corresponding to the major bands accounted for 30% of the total eubacterial cell count at the steady state. These results indicate that this bacterium is a key microorganism in the biodegradation process.

Construction of a stable microbial community with high cellulose-degradation ability.

We bred a microbial community capable of degrading rice straw with high efficiency. The microbial community degraded more than 60% of rice straw within 4 days at 50 degrees C. The high stability of the community's degradation ability was demonstrated by its tolerance of being subcultured several times in medium with/without cellulosic material, being heated to 95 degrees C, and freezing at -80 degrees C. The community degraded both nonsterilized and sterilized substrate; and its degradation ability was not affected by pH changes in the medium (initial pH 5-9). PCR-denaturing gradient gel electrophoresis (DGGE) analyses based on 16S rDNA fragments showed that the community structure remained constant after multiple subcultures extending over 2 years. DNA sequence analyses of DGGE bands indicated the coexistence of both aerobic and anaerobic bacteria in the community.

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor.

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.

Characterization of an extended-spectrum class C beta-lactamase of Citrobacter freundii.

Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino beta-lactams such as ceftazidime and aztreonam. This drug resistance was due to an extended-spectrum class C beta-lactamase encoded by chromosomal gene(s). The GC3 beta-lactamase showed high amino acid sequence homology to a known C. freundii beta-lactamase, i.e., 346 of 361 amino acids were identical with those of C. freundii GN346 beta-lactamase (Tsukamoto, K. et al, Eur. J. Biochem. 188, 15-22, 1990). Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended-spectrum resistance in class C beta-lactamases (Haruta, S. et al, Microbiol. Immunol. 42, 165-169, 1998). However, Asp198 was eliminated as a cause of the extended-spectrum resistance by the substitution of Asn for Asp198. Subsequent investigation suggested that the moderate resistance to oxyimino beta-lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active-site. Some or all of the replacements are assumed to delicately modify the active-site configuration. The GC3 beta-lactamase is the first example of an extended-spectrum class C beta-lactamase in which mutations are independent of the omega loop.

Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase.

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.

Evaluation of absorption kinetics of orally administered theophylline in rats based on gastrointestinal transit monitoring by gamma scintigraphy.

The gastrointestinal (GI) transit and absorption of orally administered theophylline, a highly absorbable drug without presystemic elimination, were investigated under fasted and fed conditions using three rats in a crossover study. To evaluate the GI transit rate for each segment in vivo, a noninvasive technique, gamma scintigraphy, was employed using a nonabsorbable compound, (99m)Tc-labeled diethylenetriamine pentaacetic acid (DTPA). Using a gamma scintigraphic technique it is possible to simultaneously evaluate the GI transit and absorption of orally administered drug in the same individual. Theophylline was simultaneously administered along with [(99m)Tc]DTPA to animals in the fasted and fed states. Each GI transit pattern, simulated using the GI transit-kinetic model with a lag time factor, was well fitted to the experimental data. Gastric emptying rate varied in each study, even under the same experimental condition. The GI transit pattern for each segment was highly variable, especially in animals in the fed state. This inconsistency in transit pattern was mainly due to the variability in gastric emptying, which was much slower in animals in the fed compared with the fasted state. However, in spite of a large variability of GI transit kinetics, the plasma concentration-time curves of theophylline were well predicted by the GI transit-absorption model using the individual GI transit parameters obtained in the study. The absorption rate of theophylline was considerably reduced in animals in the fed state, because of the reduction of gastric emptying rate. Analysis using GI transit-absorption model and gamma scintigraphic technique made it possible to estimate the variable absorption kinetics regulated by GI transit with huge variability.

Denaturing gradient gel electrophoresis analyses of microbial community from field-scale composter.

The diversity of microbial community during the decomposition of waste in a field-scale composter (Hazaka system) was investigated by denaturing gradient gel electrophoresis (DGGE). The composter operates at a high temperature through a self-heating system, creating a thermophilic (60-76 degrees C) stage during the initial phase and a mesophilic (45 degrees C) stage towards the later phase of the composting period. The pH of the system (pH 7.75-8.10) did not vary significantly during the process while moisture content was reduced from 48.8% to 25.1%. DGGE and 16S rDNA analyses showed that the following genera were found throughout the process: Propionibacterium sp., Methylobacterium sp., Pseudomonas sp., and Bradyrhizobium sp. Different Bacillus spp. thrive at the thermophilic or the mesophilic stage while Clostridium sp. was only found at the initial phase of the process. Staphylococcus sp. and Caulobacter sp. or Brevundimonas sp. existed during the later phase of the composting period.

Analyses of microbial community within a composter operated using household garbage with special reference to the addition of soybean oil.

A commercially available composter was operated using fixed composition of garbage with or without the addition of soybean oil. The composter was operated without adding seed microorganisms or bulking materials. Microflora within the composter were analyzed by denaturing gradient gel electrophoresis (DGGE) in the case of oil addition, or by 16/18 S rRNA gene sequencing of the isolated microorganisms in the case of no oil addition. The results showed that, irrespective of the addition of oil, the bacteria identified were all gram positive, and that lactobacilli seemed to be the key microorganisms. Based on the results, suitable microflora for use in a household composter are discussed.

Characterization of a microorganism isolated from the effluent of hydrogen fermentation by microflora.

The hydrogen production yield from glucose by an isolate was investigated and compared to that by microflora. The isolate, Thermoanaerobacterium thermosaccharolyticum KU001, from the microflora demonstrated approximately 2.4 mol/mol-glucose of hydrogen production with acetate/butyrate formation in an artificial medium. The fermentation pattern was similar to that observed for the hydrogen fermentation of wastewater by the microflora. A PCR-DGGE analysis of the bacterial 16S rDNA detected T. thermosaccharolyticum in the microflora with strong intensity of the characteristic 16S rDNA band, although the microflora was enriched from an artificial medium. These results imply that T. thermosaccharolyticum could be a predominant species of the microflora that is involved in hydrogen-producing acetate/butyrate fermentation. The nitrogen source in the medium affected the carbohydrate metabolism of KU001, and caused a change in hydrogen yield.

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost.

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.

X-linked dilated cardiomyopathy with a large hot-spot deletion in the dystrophin gene.

A 36-year-old Japanese man was hospitalized with coughing and exertional dyspnea (NYHA class I). He was diagnosed as having congestive heart failure, and was treated with diuretics and a beta-adrenergic blocking agent. He responded well to the treatment and his symptoms completely disappeared within a few days. Based on his clinical, laboratory, and molecular genetic findings, he was diagnosed as having X-linked dilated cardiomyopathy (XLDCM). He was found to have a large deletion in the dystrophin gene, involving exons 45-55. This is the first report on a Japanese XLDCM patient with a mutation in the central hot-spot region of this gene.

Usefulness of the subendocardial myocardial velocity gradient in low-dose dobutamine stress echocardiography.

The subendocardial side of myocardium makes a major contribution to left ventricular (LV) contraction and is very susceptible to ischemia. In this study we sought to quantify regional wall motion during low-dose dobutamine stress echocardiography (DSE) by using the myocardial velocity gradient (MVG) derived from tissue Doppler imaging (TDI). We then compared the usefulness of subendocardial MVG with that of transmural MVG in detecting subtle wall motion abnormalities. Fourteen patients (single vessel disease = 6; normal coronary arteries = 8) underwent low-dose DSE (10 microg/ kg per min). M-Mode TDI of the LV posterior wall was recorded using a Toshiba SSA-380A combined with custom computer software, and analyzed for both subendocardial and transmural MVG. Visual estimation and transmural MVG failed to clearly demonstrate the differing responses between the nonischemic (systole: 3.0 +/- 0.8/s to 4.9 +/- 1.9/s, not significant; diastole: -4.3 +/- 1.3/s to -5.7 +/- 1.4/s, not significant; mean +/- SD, P versus ischemic segments) and ischemic (systole: 3.3 +/- 1.2/s to 3.8 +/- 1.0/s; diastole: -5.4 +/- 2.0/s to -5.3 +/- 1.1/s) segments during low-dose DSE. Subendocardial MVG demonstrated a significant change in the nonischemic segments (systole: 4.1 +/- 1.0/s to 7.7 +/- 2.2/s, P = 0.012; diastole: -6.5 +/-1.8/s to -11.3 +/- 2.2/s, P = 0.001), whereas the response remained unchanged in the ischemic segments (systole: 4.6 +/-2.4/s to 4.8 +/- 1.2/s; diastole: -7.0 +/- 1.9/s to -7.3 +/- 1.1/s). Subendocardial MVG, particularly diastolic subendocardial MVG, may serve as a useful indicator of subtle ischemic changes in wall motion induced by low-dose DSE.