Isolation and characterization of an ether-type polyurethane-degrading micro-organism and analysis of degradation mechanism by Alternaria sp.

AIMS: To degrade ether-type polyurethane (ether-PUR), ether-PUR-degrading micro-organism was isolated. Moreover, ether-PUR-degrading mechanisms were analysed using model compounds of ether-PUR. METHODS AND RESULTS: A fungus designated as strain PURDK2, capable of changing the configuration of ether-PUR, has been isolated. This isolated fungus was identified as Alternaria sp. Using a scanning electron microscope, the grid structure of ether-PUR was shown to be melted and disrupted by the fungus. The degradation of ether-PUR by the fungus was analysed, and the ether-PUR was degraded by the fungus by about 27.5%. To analyse the urethane-bond degradation by the fungus, a degraded product of ethylphenylcarbamate was analysed using GC/MS. Aniline and ethanol were detected by degradation with the supernatant, indicating that the fungus secreted urethane-bond-degrading enzyme(s). PURDK2 also degraded urea bonds when diphenylmethane-4,4'-dibutylurea was used as a substrate. CONCLUSIONS: The enzyme(s) from PURDK2 degraded urethane and urea bonds to convert the high molecular weight structure of ether-PUR to small molecules; and then the fungus seems to use the small molecules as an energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: Ether-PUR-degrading fungus, strain PURDK2, was isolated, and the urethane- and urea-bonds-degrading enzymes from strain PURDK2 could contribute to the material recycling of ether-PUR.

Nasal neuroendocrine carcinoma in a free-living Japanese raccoon dog (Nyctereutes procyonoides viverrinus).

Neuroendocrine carcinoma was diagnosed in the left nasal cavity of a free-living Japanese raccoon dog (Nyctereutes procyonoides viverrinus). Microscopically, the tumour consisted of sheets of anaplastic cells separated by narrow zones of fibrovascular stroma. The neoplastic cells had varying numbers of cytoplasmic granules stained by the Grimelius method. Immunohistochemically, the neoplastic cells were variably labelled for cytokeratin AE1/AE3, vimentin, chromogranin A and S-100. Ultrastructurally, some of the neoplastic cells had cytoplasmic membrane-bound dense-core granules of approximate diameter 140-240nm. The tumour had infiltrated the cerebrum and metastasized to the pituitary gland, mandibular and pulmonary lymph nodes, lungs, thyroid gland and adrenal glands.

Influence of Nonenzymatic Glycation in Dentinal Collagen on Dental Caries.

Advanced glycation end-products (AGEs) are generated via nonenzymatic glycation of dentinal collagen, resulting in accumulation of AGEs in dentin tissue. Since accumulated AGEs cause crosslinking between amino acid polypeptides in the collagen molecule and modify mechanical properties of dentinal collagen, the authors assumed that there would be a significant interaction between the generation of AGEs and progression of caries in dentin. To confirm such an interaction, spectroscopic imaging analyses (i.e., nanosecond fluorescence lifetime imaging and second harmonic generation light imaging) were performed in addition to biochemical and electron microscopic analyses in the present study. Seven carious human teeth were fixed in paraformaldehyde and cut longitudinally into 1-mm sections using a low-speed diamond saw for the following analyses. In transmission electron microscopy (TEM) analysis, nondecalcified specimens were embedded in epoxy resin and sliced into thin sections for observation. For the immunohistochemical analysis, the specimens were paraffin embedded after decalcification for 2 wk and sectioned with a microtome. Resultant sections were stained with anti-AGE and anticollagen antibodies. The demineralized specimens were used for spectroscopic analyses without additional treatment. For Western blotting analysis, specimens were separated into carious and sound dentin. Each specimen was homogenized with a bead crusher and an ultrasonic homogenizer and then treated with hydrochloric acid. In carious dentin, the collagen fibers showed an amorphous structure in the TEM image, and the AGEs were localized in the areas of bacterial invasion in the immunostaining image. The total amount of AGEs in carious dentin was higher than in sound dentin in Western blotting. The ultrastructure of type I collagen and total amount of AGEs varied markedly in the dentinal caries region. The fluorescence lifetime was shorter in the carious area than that in the sound areas, indicating an increase of AGEs in the carious area. The increase of AGEs could influence the progression of dentinal caries.

Sequential synthesis of cartilage and bone marker proteins during transdifferentiation of mouse Meckel's cartilage chondrocytes in vitro.

Meckel's cartilage cells cultured in vitro undergo phenotypic transformation toward osteogenic cells. We examined whether these cells synthesize type X collagen and bone morphogenetic protein-2 (BMP-2). We also examined the results of Alcian blue staining and the expression of type I and type II collagen, osteocalcin and chondroitin sulfate proteoglycan (CSPG) during this transdifferentiation. Meckel's chondrocytes, isolated from day-17 mouse embryos, were inoculated at 1 x 10(4)/penicylinder and cultured in alpha-MEM for periods up to 4 weeks. Alcian blue staining and immunostaining of type II collagen and CSPG confirmed that, after cell culture for 2 weeks, the cartilaginous phenotype was expressed most intensely. Later in culture, chondrocytes underwent modification through the synthesis of bone-type proteins; nodule-forming small round cells showed ALPase activity and were immunoreactive for type I collagen and osteocalcin. Immunoreactivity for type X collagen was detected in the small round cells at the top of the nodules prior to calcification of the matrix, as well as in large hypertrophic cells. BMP-2 was also expressed first in similar small round cells after 3 weeks in culture, and it subsequently extended along the extracellular matrix in the calcified nodules. These results indicate that small round cells that are differentiating toward osteocyte-like cells from Meckel's chondrocytes express type X collagen and BMP-2 sequentially.

A genome-wide association study of periodontitis in a Japanese population.

Periodontitis is a multifactorial disease in which bacterial, lifestyle, and genetic factors are involved. Although previous genetic association studies identified several susceptibility genes for periodontitis in European populations, there is little information for Asian populations. Here, we conducted a genome-wide association study and a replication study consisting of 2,760 Japanese periodontitis patients and 15,158 Japanese controls. Although single-nucleotide polymorphisms that surpassed a stringent genome-wide significance threshold (P < 5 × 10(-8)) were not identified, we found 2 suggestive loci for periodontitis: KCNQ5 on chromosome 6q13 (rs9446777, P = 4.83 × 10(-6), odds ratio = 0.82) and GPR141-NME8 at chromosome 7p14.1 (rs2392510, P = 4.17 × 10(-6), odds ratio = 0.87). A stratified analysis indicated that the GPR141-NME8 locus had a strong genetic effect on the susceptibility to generalized periodontitis in Japanese individuals with a history of smoking. In conclusion, this study identified 2 suggestive loci for periodontitis in a Japanese population. This study should contribute to a further understanding of genetic factors for enhanced susceptibility to periodontitis.

Differential activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in zones of the adrenal cortex.

Cholesterol metabolism and steroidogenesis in the outer (zona fasciculata/glomerulosa) and inner (zona reticularis) zones of the adrenal cortex were examined in the guinea pig. It is known from previous studies that the content of cholesterol in the inner zone is considerably lower than that in the outer zone, although basal low density lipoprotein (LDL) receptor activity is similar in the two zones. To further explore cholesterol metabolism in the guinea pig adrenal cortex, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis, has been examined for which this paper forms the initial report. It was found that the basal specific activity of HMG-CoA reductase was similar in the outer and inner adrenocortical zones (approximately 230 pmol mevalonate formed/min X mg microsomal protein). The administration of ACTH caused 4- and 5-fold increases in HMG-CoA reductase activity in the outer and inner zones, respectively. In fact, the increase in HMG-CoA reductase activity with ACTH treatment was always greater for the inner zone than for the outer zone. This is in contrast to LDL receptor activity, which does not increase in the inner zone as it does in the outer zone with ACTH treatment. When dexamethasone was administered, HMG-CoA reductase activity decreased in the outer zone by about 50%, while there was no change in reductase activity in the inner zone. The latter finding is similar to what happens with LDL receptor activity during dexamethasone administration. Why suppression of endogenous ACTH had no effect on HMG-CoA reductase activity in the inner zone while exogenous ACTH administration caused a marked increase in enzyme activity is not clear, but may be related to phosphorylation/dephosphorylation mechanisms. Based on the use of sodium fluoride in solutions to block HMG-CoA reductase phosphatase, evidence is presented which indicates that a pharmacological dose of ACTH alters the phosphorylation/dephosphorylation status of HMG-CoA reductase in the inner adrenocortical zone, but not in the outer cortical zone.

Binding of propranolol and gentamicin to small unilamellar phospholipid vesicles. Contribution of ionic and hydrophobic forces.

Binding of propranolol and gentamicin to small unilamellar phospholipid vesicles having different surface charges was studied at pH 4.4 using an ultra-centrifugation method, and the results were analyzed by an equation describing the Langmuir adsorption isotherms. Gentamicin, a polycationic drug, bound to negatively-charged small unilamellar vesicles composed of 60% phosphatidylcholine and 40% of either phosphatidylinositol, phosphatidylglycerol or phosphatidylserine in a manner consistent with a single class of binding sites but did not bind at all to small unilamellar vesicles of phosphatidylcholine alone. In contrast, propranolol bound readily to both neutral and negatively-charged liposomes in a manner consistent with two types of binding sites. Based on the binding parameters calculated from replots, it is suggested that the high-affinity site is probably at the surface of the liposome and that ionic forces are primarily responsible for this binding. The low-affinity, high-capacity binding site for propranolol was demonstrated with both neutral and negatively-charged liposomes and appeared to be independent of the surface charge. Gentamicin, which is not hydrophobic, did not bind to the low-affinity site. It is hypothesized that hydrophobic interactions are the driving force for propranolol binding to the low-affinity site which may be the interior of the lipid bilayer.

The severity of periodontal disease is associated with the development of glucose intolerance in non-diabetics: the Hisayama study.

Inflammation is hypothesized to play a significant role in the development of type 2 diabetes; however, reports on clinical inflammatory conditions are limited. Studies have suggested that periodontitis affects glucose control in diabetics. This community-based study examined the relationship between periodontitis and glucose tolerance status, including changes in status. The relationship between periodontal condition and the results of a 75-g oral glucose tolerance test was examined in 961 adults in 1998. Deep pockets (mean pocket depth > 2.0 mm) were significantly associated with impaired glucose tolerance and with diabetes as compared with shallow pockets (< 1.3 mm). In the subgroup with normal glucose tolerance 10 years previously, subjects who subsequently developed impaired glucose tolerance were significantly more likely to have deep pockets. Deep pockets were closely related to current glucose tolerance status and the development of glucose intolerance.

Immunocytochemical expression of type I and type II collagens by rat Meckel's chondrocytes in culture during phenotypic transformation.

In culture, chondrocytes of Meckel's cartilage can differentiate further to become bone-type collagen-synthesizing cells. Here, the replacement of type II collagen by type I collagen, accompanying expression of the osteocytic phenotype, was analysed by double immunofluorescence staining, histochemistry and electron microscopy. After 1 week in culture, formation of a toluidine blue-positive matrix, demonstrating the synthesis of cartilaginous proteoglycans, and the expression of type II collagen were detected. After 2 weeks, immunoreactivity specific for type II collagen was detected along the cartilaginous areas of the nodules, and type I collagen appeared in association with the immunopositive extracellular matrix around spindle-shaped cells. Electron microscopy revealed that the extracellular matrix at this stage was composed of homogeneous fine fibrils of type II collagen and thick cross-banded bundles of type I collagen: there was also continuity between the type I and II collagens. Double immunofluorescence staining of 3 week-old cultures revealed that type II collagen had been replaced by type I which was synthesized by small round cells that appeared at the top of the nodules. With further passage of time in culture, the distribution of type I collagen expanded further towards the peripheral areas from the central areas of the nodules. The present combination of ultrastructural analysis and double immunofluorescence staining shows that the transition from synthesis of cartilage-specific type II collagen to expression of type I collagen occurred sequentially in spindle-shaped cells located at the top of nodules in conjunction with the further differentiation of Meckel's cartilage cells.

[Prevention and control of nosocomial infection caused by methicillin resistant Staphylococcus aureus in premature infant ward--prevention effect of "povidone iodine solution" wipe of neonatal skin].

In January 1983, a number of premature infants under management in the premature infants' unit of our hospital were found to have bacteremia due to Staphylococcus aureus. By the end of February of the same year, 4 of these infants, who had been treated in the same unit, developed impetigo. The S. aureus responsible for this condition was classified as type IV by a coagulase typing. In a subsequent antimicrobial susceptibility test using the disk diffusion method, this microorganism was found to be resistant to methicillin, erythromycin and lincomycin, and to be susceptible to tetracycline, chloramphenicol and cefmetazole, indicating that it was a methicillin resistant S. aureus (MRSA). Because the result from the coagulase typing agreed with the antimicrobial susceptibility pattern in all cases, we concluded that these cases represented nosocomial infection with MRSA. The source and route of the infection were investigated, and measures taken to prevent bacterial spread from carriers and to keep instruments and environments clean. Although the source of infection was not identified. Then, we tried wiping the body surface of the premature infants with an Isodine solution (10% PVP-I, 1:100 dilution) in order to prevent colonization of the microorganism on the body surface. With this application+, MRSA was no longer detected from the body surface of the premature infants, and no additional MRSA infection occurred in the premature infants' unit. Data collected for premature infants' managed at our hospital in the subsequent 6 years allows us to conclude that MRSA infection can be almost completely controlled by frequent surveys of carriers and appropriate body surface wiping with Isodine solution.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of massive hepatoma which responded to SMANCS/Lipiodol regimen with intra-arterial infusion].

Transcatheter arterial chemotherapy (SMANCS/lipiodol) was applied to massive hepatoma, which had a high AFP 213,000 ng/ml, A-P shunt, tumor thrombosis and metastatic lung cancer. After 3 months, the AFP value reduced to 18 ng/ml, massive hepatoma and the A-P shunt disappeared, but AFP-negative nodular hepatoma recurred around initial hepatoma. Each time, we injected SMANCS/lipiodol to the recurring hepatoma. The therapy in the initial stage was not so effective. The portal vein was not observed in the initial stage, but appeared after the second dosage. Metastatic lung cancer was declining in the initial dosage and 23 months later disappeared after the third dosage. The massive hepatoma occupied entirely the rt. lobe of the liver. The patient lived for 4 years, had total admission periods of 190 days and could return to life in society. In this case, we considered that transcatheter arterial chemotherapy (SMANCS/lipiodol) had remarkable effects.

In-beam Mössbauer spectroscopy of (57)Fe/(57)Mn in MgO and NaF at Heavy-Ion Medical Accelerator in Chiba.

Development of efficient ion supply of (58)Fe from (58)Fe(C5H5)2, and quick switching between therapy and material science at the Heavy-Ion Medical Accelerator in Chiba realized a new (57)Mn in-beam emission Mössbauer spectroscopy measurement system. Application to simple binary chemical compounds, MgO and NaF, proved the usefulness of the system to probe chemical and physical behaviors of trace impurities in solids. Annealing of lattice defects produced by the implantation and ß-decay of (57)Mn and/or γ-ray emission recoil was observed by a local probe.

Heating properties of the needle type applicator made of shape memory alloy by 3-D anatomical human head model.

Since the human brain is protected by the skull, it is not easy to non-invasively heat deep brain tumors with electromagnetic energy for hyperthermia treatments. Generally, needle type applicators were used in clinical practice to heat brain tumors. To expand the heating area of needle type applicators, we have developed a new type of needle made of a shape memory alloy (SMA). In this paper, heating properties of the proposed SMA needle type applicator were discussed. Here, in order to apply the SMA needle type applicator clinically. First, we constructed an anatomical 3-D FEM model from MRI and X-ray CT images using 3D-CAD software. Second, we estimated electric and temperature distributions to confirm the SMA needle type applicator using the FEM soft were JMAG-Studio. From these results, it was confirmed that the proposed method can expand the heating area and control the heating of various sizes of brain tumors.

Finite element analysis of the needle type applicator made of shape memory alloy.

In this paper, we propose a new heating method in which we use shape memory alloy (SMA) in a needle type applicator for brain tumor hyperthermia. In order to expand the heating area of a needle type applicator and to control the heating pattern for various sizes of tumors, some kinds of SMA needle type applicators were developed. To apply the proposed heating method safely to clinical hyperthermia, it is necessary to make appropriate thermal distribution to the region of the brain tumor. However, it is not easy to predict the three dimensional temperature distribution during the human brain tumor hyperthermia. Therefore, we estimated the temperature distribution inside the agar phantom by the finite element method (FEM). Here, first, the computer simulation results of temperature distributions under the different heating times are discussed. Second, a comparison of the heating properties obtained by using the needle type electrodes made of different shaped SMA is discussed. From these results, it is confirmed that the proposed heating method can expand the heating area and control the heating pattern for the various sizes of brain tumors.

Relationship between obesity, glucose tolerance, and periodontal disease in Japanese women: the Hisayama study.

BACKGROUND: Recent studies have reported a relationship between obesity and periodontal disease. Obesity is the strongest risk factor for type 2 diabetes, which is, in turn, a risk factor for periodontal disease. An oral glucose tolerance test is necessary to diagnose diabetes; however, no study has examined the relationship between obesity and periodontal disease by taking oral glucose tolerance test results into consideration. METHODS: In all, 584 Japanese women aged between 40 and 79 years old, with at least 10 teeth, underwent health examinations. Body mass index, waist-hip ratio, body fat, and oral glucose tolerance test results were used as independent variables with known risk factors for periodontal disease. Mean probing pocket depth and mean attachment loss were used as the dependent variables. RESULTS: In all of the analyses, body mass index, body fat, and waist--hip ratio were significantly associated with the highest quintile of mean probing pocket depth, even when adjusted for oral glucose tolerance test results. In the multivariate analysis, the subjects with the highest quartile of body mass index had a significantly higher odds ratio (OR) for the highest quintile of mean probing pocket depth [OR, 4.3; 95% confidence interval (CI), 2.1--8.9; p<0.001], whereas neither impaired glucose tolerance nor diabetes were significantly associated with deep pockets. The relationships between the obesity indexes and mean attachment loss did not reach statistical significance. CONCLUSION: Obesity was associated with deep pockets in Japanese women, even after adjusting for oral glucose tolerance test.

[Visible-light cured resin. Selection of reducing agent amine, its appropriate amount and effect on physical properties].

Studies were done to improve the physical properties, especially degree of conversion of visible-light cured resin (VLCR), and accordingly to lower its water sorption and solubility in water. Trial products of VLCR were prepared using various kinds of amine, reducing agent, and the photoinitiator to be mixed with cyclophosphazene monomer, 4 PN-(TF)1-(EMA)7. As amines ethyl-p-dimethylaminobenzoate (DMAB), methacryloxyethyl-p- dimethylaminobenzoate (DMAB-EMA) and methacryloxyglycidyl-p-dimethylaminobenzoate (DMAB-GMA) were used. Of their set products immersed in water and MeOH, their water sorption, solubility in water, MeOH sorption and solubility in MeOH were examined. The resin with DMAB-EMA used was preferable, showing comparatively small solubility. Next, VLCR, with various amounts of DMAB-EMA mixed, 0.5-5.0 in mol ratio to photosensitizer (CQ + DB), and of their set products water sorption, solubility in water, MeOH sorption, solubility in MeOH, THF sorption and their mechanical properties after immersion in water were examined. Immersion of the resin products in water and MeOH for 30 days, lowered the solubility in water and MeOH to a minimum at the mixing ratio of 3.0 in mol ratio. Solubility in MeOH (HPLC) became minimum at 2.0 in mol ratio, and at less than 2.0, the photosensitizer and monomer were dissolved, while at more than 2.0, the photosensitizer and DMAB-EMA were dissolved. THF sorption decreased in accordance with the increase in the mixed amount of DMAB-EMA, became almost constant at more than 3.0 in mol ratio. The compressive strength of set product after immersion in water for 7 days increased in accordance with the increase of the mixed amount of DMAB-EMA, while the transverse strength also increased up to 2.0 in mol ratio. The optimum reducing agent to be mixed with VLCR was DMAB-EMA, and the mixing ratio should be 2.0-3.0 in mol ratio to photosensitizer.

Epidemiological observations on bluetongue in sheep and cattle in Japan.

Bluetongue (BT) first occurred in Japan between late August and October 1994 in 23 cattle in three prefectures of the northern Kanto region, and between the end of October and mid-November in 23 Suffolk sheep in the same region. The affected cattle had fever, deglutitive difficulty, hyper-salivation, facial oedema, scabbing of the corner of the mouth and dysphagia. The BT virus (BTV) was isolated from blood cells of the affected sheep. Surveillance for BTV antibody conducted by prefectures in the affected region has detected seroconversion to BTV in some prefectures every year thereafter. In the autumn of 2001, again in the northern Kanto region, 45 sheep developed BT, and BTV was isolated. It is considered important that Japan has imported numerous cattle from Australia, the United States of America (USA), and New Zealand every year. In particular, BTV was isolated from cattle imported from the USA during quarantine although some of the serotypes isolated are not present in the USA. Furthermore, BTV is not present in New Zealand. The third RNA segment encoding the serogroup-specific VP3 protein of Japanese BTV isolates and reverse transcriptase-polymerase chain reaction (RT-PCR) positive blood cells was amplified by RT-PCR. Molecular phylogenetic analysis of the third RNA segment based on the sequence homology of the PCR products led to the classification of Japanese BTV isolates into two major groups.

[Historical investigation of dentifrice in China].

A herbal literature survey was carried out on data concerning historical pharmacognostical changes of "dentifrice" in China and how diseases of the teeth and gums had been treated there in ancient times. It had been considered to be a matter of utmost importance that to prevent teeth from decaying, only the brushing of teeth with a toothbrush was necessary. Over time, various tooth agents have been found to treat oral diseases the teeth and gums. Glycyrrhizae Radix, Ginseng Radix, Scutellariae Radix, Menthae Herbal, and salt were widely used materials. Investigations from all approaches are being carried out to develop remedies for oral diseases, including Kampo medicine and the pharmacological effects of numerous crude drugs. When tracing the pharmacognostical changes of dentifrice in ancient China, we felt wonder at and admiration for the abundance of clinical experiences described in the old herbal and medical literature we researched.

Mechanism of cationic amphiphilic drug inhibition of purified lysosomal phospholipase A1.

Cationic amphiphilic drugs like chlorpromazine, propranolol, and chloroquine inhibit lysosomal phospholipase A in vitro. Some workers have proposed that cationic amphiphilic drugs inhibit the activity of phospholipase A1 by forming substrate-drug complexes which cannot be degraded while others have reported competitive inhibition implying drug effects on the enzyme. To analyze the mechanism of inhibition, we examined the binding ability of these drugs to unilamellar vesicles of dioleoylphosphatidylcholine and correlated these results with a detailed kinetic analysis of phospholipase A. Chlorpromazine and propranolol bound to small unilamellar liposomes of dioleoylphosphatidylcholine substrate in a positive cooperative way consistent with two binding sites: a high-affinity site with low capacity and a low-affinity site with high capacity. The affinity of chlorpromazine for the high-affinity site was 2 times greater than that of propranolol (KA = 13 807 +/- 1722 vs. 8481 +/- 1078 M-1), and the saturation number for chlorpromazine was 3 times greater than for propranolol (N = 0.20 +/- 0.004 vs. 0.07 +/- 0.02 mol of drug/mol of phosphatidylcholine). Chloroquine did not bind to unilamellar liposomes of dioleoylphosphatidylcholine. We carried out detailed kinetic studies using purified lysosomal phospholipase A1 from rat liver. In the case of chloroquine inhibition, the Lineweaver-Burk double-reciprocal plots showed straight lines, but the slope replots were curved, indicating the formation of complexes having 2 mol of chloroquine/mol of enzyme (EI2 complexes). Thus, chloroquine is a competitive inhibitor which forms EI2 complexes with phospholipase A1. However, in the case of chlorpromazine and propranolol, the observed kinetic data do not fit to the same equilibrium used for the case of chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)