Biomedical subjects

Y Sekiguchi

Publications and source records attributed to Y Sekiguchi.

At least 19 recordsLinked to original sources

PubMed2005

Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors.

We developed a rapid and simple method for rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems. The method relies on the sequence-specific scission of 16S rRNA with ribonuclease H (RNase H) and oligonucleotides that specifically hybridize with targeted rRNA molecules. RNAs from a complex community were first mixed with an oligonucleotide and were subsequently digested with RNase H to achieve sequence-dependent rRNA cleavage at the hybridization site. For the quantitative detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel-electrophoresis, which separated and quantified cleaved and intact rRNA fragments. This method enabled the quantitative detection of microbes in a complex microbial community by a relatively simple and fast experimental procedure. We then applied the cleavage method to actual anaerobic microbial communities such as digested sewage sludge and UASB sludges. The results demonstrated that the present method was fully applicable to anaerobic digestor ecosystems containing complex anaerobic microorganisms.

Bioreactors↗

PubMed2002

Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges.

The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e. who are present, where they are located, and what they are doing there. To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis. In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes. The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries. 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules. Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters. This resulted in the detection of two types of uncultured cells in specific locations inside the granules. Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions. In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium. In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40%. In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

Bacteria, Anaerobic↗

PubMed2002

Pilot-plant study on anaerobic treatment of a lipid- and protein-rich food industrial wastewater by a thermophilic multi-staged UASB reactor.

An on-site pilot-scale experiment was conducted to investigate the performance of a multi-staged UASB (MS-UASB) reactor by feeding with a food processing wastewater containing high strength of lipid and protein. The reactor was operated at a thermophilic condition (55 degrees C) for a period of 600 days. The reactor finally achieved 50 kgCOD.m(-3) d(-1) with a soluble COD removal of 90% (based on the influent total COD versus the effluent filtered COD), while the overall COD removal (based on the effluent COD-total) as considerably unsatisfactory at around only 60-70%. The presence of high strength of lipid and protein along with high concentration of Mg and Ca ions in the raw wastewater caused a severe scum and/or insolubilized substance formation within the UASB sludge bed, resulting in hindering the contact efficiency between substrate and sludge. The replacement of active microbial granules in the sludge bed with the insolubilized protein and lipid brought about deterioration of sludge methanogenic activity.

Bacteria, Anaerobic↗

PubMed2001

Brain-derived gangliosides suppress the chronic relapsing-remitting experimental autoimmune encephalomyelitis in NOD mice induced with myelin oligodendrocyte glycoprotein peptide.

Chronic relapsing-remitting experimental autoimmune encephalomyelitis (CREAE) induced with myelin oligodendrocyte glycoprotein peptides 35-55 (MOG(35-55)) in NOD mice was successfully treated with brain-derived gangliosides (GA). The GA treatment suppressed the development and severity of CREAE, both clinically and histologically. Spleen cells from the GA-treated mice displayed markedly inhibited levels of MOG(35-55) specific proliferation and interferon-gamma production. Delayed-type hypersensitivity reactions to MOG(35-55) were suppressed by the GA treatment. GA modulate various T cell effector functions in CREAE and may be an effective therapeutic agent for autoimmune demyelinating diseases such as multiple sclerosis.

Amino Acid Sequence↗

PubMed2001

Plasma thrombin-antithrombin III complex is associated with the severity of experimental autoimmune encephalomyelitis.

Previous studies have shown that activation of blood coagulation and fibrin depositions around CNS vessels are observed in animals with experimental autoimmune encephalomyelitis (EAE), which provides an animal model for human autoimmune demyelinating disorders. We examined the values of peripheral blood fibrinogen, thrombin-antithrombin III complex (TAT), fibrinolytic activity, and fibrin degradation products in Lewis rats with EAE to elucidate the role of the blood coagulation-fibrinolysis system in EAE. Plasma TAT values increased immediately prior to development of symptoms, and decreased according to the improvement of symptoms. There was significant correlation between TAT values and clinical scores of EAE; other markers were not correlated with the symptoms of EAE. These results suggest that plasma TAT levels are sensitive markers of the severity of EAE, and may be useful clinical indicators for the severity of human autoimmune demyelinating disorders.

Animals↗

PubMed2001

Recent advances in methane fermentation technology.

In the past two decades, a number of biotechnologies for anaerobic (methanogenic) wastewater treatment have been created, and practical applications of these processes are now being extended to more recalcitrant wastewaters and to wastewaters at extreme temperatures. Our knowledge of methanogenic organic degradation associated with bioreactors is also accumulating at a rapid rate. The recent advancement of such fundamental understanding is attributed to modern molecular biology techniques applied to the study of microbial communities and to continuous challenges to the cultivation of many important but recalcitrant anaerobes in bioreactors.

Anaerobiosis↗

PubMed2001

Quinapril with high affinity to tissue angiotensin-converting enzyme reduces restenosis after percutaneous transcatheter coronary intervention.

Experimental studies have demonstrated that vascular injury resulted in an induction of vascular angiotensin-converting enzyme (ACE), and have suggested that inhibition of vascular ACE might be important in the prevention of restenosis. The present study aimed to determine the effect of quinapril, an ACE inhibitor with high affinity to tissue ACE, on restenosis following coronary intervention. The design of this study was a prospective, randomized, open, and non-placebo controlled trial. Patients with ischemic heart disease were enrolled after successful percutaneous transluminal coronary angioplasty or stent implantation at 7 participating institutions. Two hundred and fifty-three patients with 294 lesions were randomly assigned to the quinapril (10-20 mg per day) group or control group. Administration of quinapril was continued for 3-6 months of the follow-up. Quantitative coronary angiography was performed before and after angioplasty and at follow-up. Core laboratory measurements were performed independently and blinded. Follow-up angiography was performed in 108 patients with 124 lesions in the quinapril group and in 107 patients with 130 lesions in the control group. The baseline characteristics and findings of angioplasty showed no significant differences between the two groups. However, in the quinapril group, restenosis per patient and per lesion was significantly lower (34.3% vs. 47.7%, p < 0.05 and 30.6% vs. 43.8%, p < 0.05). Multivariable analysis revealed that administration of quinapril independently contributed to reducing the restenosis per patient and per lesion (odds ratio, 0.73; 95% confidence interval, 0.54-0.99 and odds ratio, 0.75; 95% confidence interval, 0.57-0.99). In conclusion, quinapril significantly reduces restenosis following coronary intervention.

Aged↗

PubMed2001

In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I.

We previously showed that very thin filamentous bacteria affiliated with the division green non-sulfur bacteria were abundant in the outermost layer of thermophilic methanogenic sludge granules fed with sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol. 65:1280-1288, 1999). Further 16S ribosomal DNA (rDNA) cloning-based analysis revealed that the microbes were classified within a unique clade, green non-sulfur bacteria (GNSB) subdivision I, which contains a number of 16S rDNA clone sequences from various environmental samples but no cultured representatives. To investigate their function in the community and physiological traits, we attempted to isolate the yet-to-be-cultured microbes from the original granular sludge. The first attempt at isolation from the granules was, however, not successful. In the other thermophilic reactor that had been treating fried soybean curd-manufacturing wastewater, we found filamentous microorganisms to outgrow, resulting in the formation of projection-like structures on the surface of granules, making the granules look like sea urchins. 16S rDNA-cloning analysis combined with fluorescent in situ hybridization revealed that the projections were comprised of the uncultured filamentous cells affiliated with the GNSB subdivision I and Methanothermobacter-like cells and the very ends of the projections were comprised solely of the filamentous cells. By using the tip of the projection as the inoculum for primary enrichment, a thermophilic, strictly anaerobic, filamentous bacterium, designated strain UNI-1, was successfully isolated with a medium supplemented with sucrose and yeast extract. The strain was a very slow growing bacterium which is capable of utilizing only a limited range of carbohydrates in the presence of yeast extract and produced hydrogen from these substrates. The growth was found to be significantly stimulated when the strain was cocultured with a hydrogen-utilizing methanogen, Methanothermobacter thermautotrophicus, suggesting that the strain is a sugar-fermenting bacterium, the growth of which is dependent on hydrogen consumers in the granules.

Animals↗

PubMed2001

Flow-through chemiluminescence sensor using immobilized histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007 and peroxidase for selective determination of histamine.

A flow sensor with immobilized oxidases is proposed for the determination of histamine in fish meat. Chemiluminometric measurement of histamine was based on the luminol reaction with hydrogen peroxide produced by immobilized histamine oxidase (EC 1.4.3.-.) and peroxidase (EC 1.11.1.7.) within a flow cell. Histamine oxidase was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The oxidase and peroxidase were coimmobilized covalently on tresylated hydrophilic vinyl polymer beads and packed into transparent PTFE; the tubing was used as the flow cell. One assay for histamine was done at intervals of 2 min without carryover. The calibration curve for histamine was linear from 0.1 microM to 50 microM. The response was reproducible within 1.25% of the relative standard deviation for 115-replicate injections of 50 microM histamine. The sensor system was applied to the determination of histamine in fish meat extracts.

Amine Oxidase (Copper-Containing)↗

PubMed2000

Analysis of condensed phosphates in food products by ion chromatography with an on-line hydroxide eluent generator.

An ion chromatographic method with gradient elution using an automated eluent generator was developed for the simultaneous determination of condensed phosphates (CPs) such as orthophosphate (P1), pyrophosphate (P2), polyphosphate, trimetaphosphate and phytate in food products. The linear calibration curves for P1, P2, tripolyphosphate, and tetrapolyphosphate in the range 0.5-500 microM had a correlation factor of 0.999 or better. The precision of the method for the CP peak areas obtained with the hydroxide eluent generator was better than that obtained with potassium hydroxide eluents prepared off-line. This method was applied to the determination of CPs in food products such as ham, fish paste, and cheese extracted by trichloroacetic acid.

Chromatography, Liquid↗

PubMed2000

Sensory innervation of the lumbar dura mater passing through the sympathetic trunk in rats.

STUDY DESIGN: Sensory innervation of the lumbar dura mater passing through the sympathetic trunk was investigated by neuronal tracing and immunohistochemical methods. OBJECTIVE: To assess an anatomic basis indicating that sympathetic block in the higher lumbar region is effective for patients with low back pain. SUMMARY OF BACKGROUND DATA: Low back pain is elicited by disorder or mechanical stimulation of the lumbar dura mater. Clinically, the authors often have observed patients in whom root block at the responsible level or sympathetic block at the higher level could relieve low back pain. Therefore, there may be two different sensory routes, the segmental innervation at the same level and nonsegmental fibers from higher dorsal root ganglia. METHODS: The tracers were injected into the sympathetic trunk between L3 and L4 of rats. The lumbar dorsal root ganglia and dura mater were examined, and labeled cells were measured in size and the distribution. To establish the sensory property, the materials were processed in immunohistochemistry for calcitonin gene-related peptide. RESULTS: Many small- to medium-sized neurons were retrogradely labeled L1 and L2 dorsal root ganglia after injection into the sympathetic trunk. The anterogradely labeled fibers were found in the dura mater at L4 and L5. Some of the labeled neurons and fibers were immunoreactive for calcitonin gene-related peptide. CONCLUSION: Sensory fibers from the upper lumbar ganglia innervated the lower lumbar dura mater directly. These sensory nerves may mediate low back pain and possibly interact with sympathetic nerves.

Animals↗

PubMed2000

Anti-IL-12 antibody prevents the development and progression of multiple sclerosis-like relapsing--remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte glycoprotein peptide.

Treatment with monoclonal anti-IL-12 antibody injected on day 0, 7 and 10 after immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 in NOD mice resulted in significant suppression of the development and the severity of the chronic relapsing-remitting experimental autoimmune encephalomyelitis (EAE) both clinically and histologically. The spleen cells from anti-IL-12 antibody treated mice displayed markedly inhibited MOG35-55 specific proliferation and IFN-gamma production. MOG35-55 specific antibody production was enhanced by anti-IL-12 antibody treatment. These results suggest that IL-12 is critically involved in the pathogenesis of MOG-induced EAE and that antibody to IL-12 could be an effective therapeutic agent in the clinical treatment of autoimmune demyelinating diseases such as multiple sclerosis (MS).

Animals↗

PubMed2000

Automatic detection of rapid eye movements by discrete wavelet transform.

In order to detect rapid eye movements (REM) automatically, the Discrete Wavelet Transform was applied to each 8-s segment of electrooculogram (EOG) data for 30 min of 8 h of normal sleep. The Haar function was used as an analysing wavelet because this function is similar to the REM waveform. By shifting the phase of the analysing wavelet by pi/4 of the function, 96% of REM could be detected. The artifacts caused by body movements could be detected simultaneously by this method. Computing time required for the detection of REM was only 11 s for 30 min EOG data.

Algorithms↗

PubMed2000

Syntrophothermus lipocalidus gen. nov., sp. nov., a novel thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate.

A new anaerobic, thermophilic, syntrophic, fatty-acid-oxidizing bacterium designated strain TGB-C1T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor. The cells were slightly curved rods and were weakly motile. Spore formation was not observed. The optimal temperature for growth was around 55 degrees C and growth occurred in the range 45 to 60 degrees C. The pH range for growth was 5.8-7.5, and the optimum pH was 6.5-7.0. Crotonate was the only substrate that allowed the strain to grow in pure culture. However, in co-culture with the thermophilic, hydrogenotrophic Methanobacterium thermoautotrophicum strain delta H, the isolate could syntrophically oxidize saturated fatty acids with 4-10 carbon atoms, including isobutyrate. During the degradation of isobutyrate by the co-culture, isobutyrate was isomerized to butyrate, which was then oxidized. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate or Fe(III) as electron acceptor. The DNA base composition was 51.0 mol%. 16S rDNA sequence analysis revealed that the strain belongs to the family Syntrophomonadaceae, but it was only distantly related to other known species of beta-oxidizing syntrophs. Hence, the name Syntrophothermus lipocalidus is proposed for TGB-C1T as a new species of a new genus.

Bacterial Typing Techniques↗

PubMed2000

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge.

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.

Bacteria↗

PubMed2000

[Rebound of prostate specific antigen after discontinuation of antiandrogen therapy for benign prostatic hyperplasia].

We compared the prostate specific antigen (PSA) levels in benign prostatic hyperplasia (BPH) patients with antiandrogen chlormadinone acetate (CMA) or allylestrenol (AE) before and during the treatment. We also investigated the serial change of PSA levels before, during and after discontinuation of antiandrogen therapy. Fifty-one BPH patients with normal PSA levels were treated with CMA or AE for 16 weeks. The mean serum PSA level significantly decreased after the treatment from 1.9 +/- 1.0 ng/ml to 1.1 +/- 0.7 ng/ml (M +/- SD). We discontinued medication with informed consent and the patients were carefully monitored for another 16 weeks. Nineteen patients were followed for 32 weeks. The mean serum PSA level decreased significantly from 2.0 +/- 1.0 ng/ml to 1.1 +/- 0.5 ng/ml (M +/- SD) and recovered approximately to the pretreatment level (1.7 +/- 1.1 ng/ml) at the end of this study. We found only one patient whose PSA was slightly elevated to a subnormal range (4.3 ng/ml) after discontinuation of therapy. The other BPH patients with normal PSA levels showed no excessive increase in PSA levels beyond the normal limit after discontinuation of antiandrogen therapy compared with the pretreatment baseline. In conclusion, BPH patients with a marked increase in PSA after discontinuation of antiandrogen therapy should be checked for prostate cancer.

Aged↗

PubMed1999

Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures.

A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus. Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei. Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA. We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus. Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive. Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function. When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced. The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect. Taken together, we suggest that Tom1 is a ubiquitin ligase. As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein. We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.

Amino Acid Sequence↗

PubMed1999

Design, synthesis, structure-activity relationships, and biological characterization of novel arylalkoxyphenylalkylamine sigma ligands as potential antipsychotic drugs.

sigma Receptor antagonists may be effective antipsychotic drugs that do not induce motor side effects caused by ingestion of classical drugs such as haloperidol. We obtained evidence that 1-(2-dipropylaminoethyl)-4-methoxy-6H-dibenzo[b,d]pyran hydrochloride 2a had selective affinity for sigma receptor over dopamine D2 receptor. This compound was designed to eliminate two bonds of apomorphine 1 to produce structural flexibility for the nitrogen atom and to bridge two benzene rings with a -CH2O- bond to maintain the planar structure. In light of the evidence, N, N-dipropyl-2-(4-methoxy-3-benzyloxylphenyl)ethylamine hydrochloride 10b was designed. Since compound 10b had eliminated a biphenyl bond of 6H-dibenzo[b,d]pyran derivative 2a, it might be more released from the rigid structure of apomorphine 1 than compound 2a. The chemical modification of compound 10b led to the discovery that N, N-dipropyl-2- [4-methoxy-3-(2-phenylethoxyl)phenyl]ethylamine hydrochloride 10g (NE- 100), the best compound among arylalkoxyphenylalkylamine derivatives 3, had a high and selective affinity for sigma receptor and had a potent activity in an animal model when the drug was given orally. We report here the design, synthesis, structure-activity relationships, and biological characterization of novel arylalkoxyphenylalkylamine derivatives 3.

Animals↗