Biomedical subjects

H Unno

Publications and source records attributed to H Unno.

At least 19 recordsLinked to original sources

PubMed2006

The survival response of Escherichia coli K12 in a natural environment.

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4 degrees C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4 degrees C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28 degrees C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28 degrees C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28 degrees C.

Bacteriological Techniques↗

PubMed2005

Neuroprotective effect of 1-aminocyclopropanecarboxylic acid in a rabbit spinal ischemia model.

BACKGROUND: Postoperative paraplegia remains a serious complication after repair of a thoracoabdominal aortic aneurysm. The release of the excitatory neurotransmitter glutamate might be responsible for ischemic neuronal damage. We investigated the effect of 1-Aminocyclopropanecarboxylic acid (ACPC) on the prevention of paraplegia in a rabbit model of spinal cord ischemia. METHODS: The infrarenal abdominal aorta was clamped in eighteen rabbits. Saline (group A), 20 mg/kg of ACPC (group B), or 10 mg/kg of ACPC (group C), was injected into the clamped aorta. The animals were neurologically evaluated by Tarlov's score. The spinal cord was obtained for histopathologic examination, including hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining method. RESULTS: Postoperative complete paraplegia with marked neuronal necrosis appeared in group A animals. Four of the six group C animals showed intact neurological function. Scattered TUNEL positive neurons were seen within areas containing necrotic cells in sections from paraplegic animals in group A. CONCLUSIONS: ACPC can prevent spinal cord injury in a rabbit model of spinal cord ischemia.

Amino Acids, Cyclic↗

PubMed2004

Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis.

To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic beta-lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.

Anti-Bacterial Agents↗

PubMed2003

Toward rational control of Escherichia coli O157:H7 by a phage cocktail.

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.

Adsorption↗

PubMed2002

Effect of dispersing oil phase on the biodegradability of a solid alkane dissolved in non-biodegradable oil.

Acinetobacter sp. CR was grown on a model oil, which consisted of an inert oil matrix of pristane with n-heneicosane dissolved in it as the sole carbon source, in a stirred-tank bioreactor. This bacterium takes up substrates from the oil phase by direct contact with the oil phase. A previously established mathematical model was applied to reveal the effect of agitation conditions on the growth and n-alkane degradation kinetics of the bacterium. Higher impeller speed resulted in both lower microbial growth and lower n-alkane degradation rate of the bacterium, although it increased the specific surface area of the oil, which was measured by a previously developed device. This result was due to the decreased number of cells adhering to the oil surface, i.e., intense agitation inhibited the adhesion of cells to the oil surface. The addition of a surfactant below a critical micelle concentration (CMC) inhibited the degradation of n-heneicosane dissolved in pristane, although the biodegradability of the substrate recovered gradually with the increase in the dose of surfactant over CMC. The results suggest that efforts to increase the specific surface area of the oil phase have the undesirable result of inhibiting oil degradation when the dominant microbial degraders take up substrates in oil by direct contact with the oil.

Acinetobacter↗

PubMed2002

Construction of self-disruptive Bacillus megaterium in response to substrate exhaustion for polyhydroxybutyrate production.

In order to establish a novel recovery system for polyhydroxyalkanoates, a self-disruptive strain of Bacillus megaterium that responds to substrate exhaustion was constructed. A gene cassette carrying the lysis system of Bacillus amyloliquefaciens phage - holin and endolysin - was inserted into the Escherichia coli- Bacillus subtilis shuttle vector pX under the control of a xylose-inducible expression system, xylR-xylA '. In this system, the expression of a target gene is induced by xylose but inhibited by glucose, which acts as an anti-inducer. B. megaterium was transformed with pX conveying the phage lysis system, which was integrated into the amyE locus of chromosomal DNA of B. megaterium by homologous recombination. The lysis system caused self-disruption of the transformant cells effectively even when expression of the lysis genes was induced during stationary phase. For the production of polyhydroxybutyrate (PHB), the transformant was grown in a medium containing glucose as a substrate in the presence of xylose. When the glucose concentration approached zero, self-disruption was spontaneously induced, releasing intracellularly accumulated PHB into the culture broth. This system realizes timely cell disruption immediately after the PHB content in the cell reaches a maximum level.

Bacillus megaterium↗

PubMed2002

Fungal contribution to in situ biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film in soil.

The contribution of fungi to the microbial degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films in soil was studied. Various traces, cavities, and grooves observed on the dented surface of PHBV films demonstrated that the degradation was a concerted effect of a microbial consortium colonizing the film surface, including fungi, bacteria, and actinomycetes. The succession of microbial consortia in the soil around the PHBV films during the degradation showed a distinctive increase in the fungal population, resulting in its dominance. Comparison of the degradation ability of microbial strains isolated from soil where PHBV films were degraded, revealed that fungi showed the highest contribution to PHBV degradation, growing very rapidly along the film surface with their high degradation ability and then expanding their hyphae in a three-dimensional manner.

Bacteria↗

PubMed2002

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.

Coliphages↗

PubMed2001

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.

Anti-Bacterial Agents↗

PubMed2001

High serum IgG4 concentrations in patients with sclerosing pancreatitis.

BACKGROUND: Sclerosing pancreatitis is a unique form of pancreatitis that is characterized by irregular narrowing of the main pancreatic duct, lymphoplasmacytic inflammation of the pancreas, and hypergammaglobulinemia and that responds to glucocorticoid treatment. Preliminary studies suggested that serum IgG4 concentrations are elevated in this disease but not in other diseases of the pancreas or biliary tract. METHODS: We measured serum IgG4 concentrations using single radial immunodiffusion and an enzyme-linked immunosorbent assay in 20 patients with sclerosing pancreatitis, 20 age- and sex-matched normal subjects, and 154 patients with pancreatic cancer, ordinary chronic pancreatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or Sjögren's syndrome. Serum concentrations of immune complexes and the IgG4 subclass of immune complexes were determined by means of an enzyme-linked immunosorbent assay with monoclonal rheumatoid factor. RESULTS: The median serum IgG4 concentration in the patients with sclerosing pancreatitis was 663 mg per deciliter (5th and 95th percentiles, 136 and 1150), as compared with 51 mg per deciliter (5th and 95th percentiles, 15 and 128) in normal subjects (P<0.001). The serum IgG4 concentrations in the other groups of patients were similar to those in the normal subjects. In patients with sclerosing pancreatitis, serum concentrations of immune complexes and the IgG4 subclass of immune complexes were significantly higher before glucocorticoid therapy than after four weeks of such therapy. Glucocorticoid therapy induced clinical remissions and significantly decreased serum concentrations of IgG4, immune complexes, and the IgG4 subclass of immune complexes. CONCLUSIONS: Patients with sclerosing pancreatitis have high serum IgG4 concentrations, providing a useful means of distinguishing this disorder from other diseases of the pancreas or biliary tract.

Adult↗

PubMed2001

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.

Adult↗

PubMed2001

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.

Journal Article↗

PubMed2000

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.

Acute Disease↗

PubMed2000

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.

Journal Article↗

PubMed2000

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.

Journal Article↗

PubMed2000

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.

Journal Article↗

PubMed2000

Effects of exercise conditioning on vital age in hyperlipidemic women.

We attempted to determine the effectiveness of exercise prescription for women with hyperlipidemia (HL), because some HL women showed no significant improvement in serum lipid and lipoprotein profiles despite the continuation of exercise conditioning. We therefore tested the hypothesis that exercise can be clinically beneficial even when there are no alterations in total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and/or triglycerides (TG). Twenty-three women with HL (aged 54.3 +/- 7.6 yr) participated in our outpatient-supervised exercise conditioning program for 90 minutes each session, twice a week for 1 year, and also did home-based exercise three days a week. Vital age (VA) is a useful index for assessing functional status, especially the risk of coronary heart disease (CHD) and is computed from 11 independent variables measured not only when a person is at rest but also during exercise. Although TC and LDLC did not change significantly after the exercise conditioning, TG decreased significantly (P < 0.05). Furthermore, when we computed the corrected VA (VAc) assuming no changes in TC, LDLC, and TG, VAc was found to decrease significantly from 64.0 +/- 9.9 yr to 58.4 +/- 9.0 yr (P < 0.05). In conclusion, these results indicate the effectiveness of exercise conditioning on risk of CHD in HL women, irrespective of alterations in the concentrations of serum lipid and lipoprotein.

Adult↗

PubMed1999

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.

Alginates↗