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.

Evaluation of soil bacterial biomass using environmental DNA extracted by slow-stirring method.

A simple and rapid method (slow-stirring method) for extracting environmental DNA (eDNA) from soils was constructed by physical mild stirring with chemical treatment. eDNA was extracted efficiently with minimal damage from various kinds of soil. The amount of eDNA and soil bacterial biomass showed a linear proportional relation [Y=(1.70x10(8))X, r2=0.96], indicating that bacterial biomass could be evaluated by quantifying levels of eDNA. Consequently, the average bacterial biomass in an agricultural field was calculated as 5.95x10(9) cells/g sample, approximately 10-100 times higher than that in non- and oil-polluted fields.

Mutational effect for stability in a conserved region of thermolysin.

AIMS: To investigate the mutational effect for the stability of thermolysin (TLN) in conserved regions. METHODS AND RESULTS: Mutational effects for stability at autodegradation sites of TLN in conserved region were studied. The bands of mutant TLN (34 kDa) on SDS-PAGE were decreased. However, those of mutant TLN cultivated with CaCl2 recovered to the same level as WT TLN. Dialysis study shows that these mutant TLN require more calcium ions than WT TLN. CONCLUSIONS: From these results, calcium affinity of mutant TLN in the conserved regions seem to become weak, subsequently mutant TLN were easily autodegraded in the case of low concentration of CaCl2. SIGNIFICANCE AND IMPACT OF THE STUDY: The autodegradation sites located in conserved regions of bacilli neutral proteases are important for the tertiary structure formation concerning the stability of the protein.

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

Identification of and hydrogen peroxide production by fecal and vaginal lactobacilli isolated from Japanese women and newborn infants.

We identified Lactobacillus isolates from Japanese women and newborn infants by a DNA-DNA hybridization method. The predominating lactobacilli were Lactobacillus crispatus and Lactobacillus gasseri in the women's vaginas and the newborns' intestines and L. gasseri and Lactobacillus fermentum in the women's intestines. All L. crispatus strains were exclusively strong H2O2 producers.

Identification of Lactobacillus species of human origin by a commercial kit, API50CHL.

The efficacy of a biochemical kit, API50CHL kit, for identification of intestinal and vaginal lactobacilli from humans was evaluated by comparing with the results of DNA-DNA hybridization assay. The results showed that in total only 52 of the 172 strains (30.2%) tested were identified correctly by the kit at species level. Especially all strains of some species, such as Lactobacillus gasseri and Lactobacillus crispatus, were misidentified as Lactobacillus acidophilus by the kit. However, the kit was found to be reliable for identification of Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus salivarius. This suggests that the exact identification of Lactobacillus isolates from human stool and vaginal specimens by API50CHL kit is difficult without the support of modern genotypic technique.

Prognostic value of visual evoked potentials (VEP) in infants with visual inattentiveness.

Visual evoked potentials elicited by strobe flash (fVEPs) were recorded in 56 infants (3 months to 15 months of age) with visual inattentiveness but without prechiasmal problems. Their visual status was reexamined one or more years later when 41 children were found to be visually competent (Group NB) and 15 were blind (Group B). We also evaluated a group of 32 age-matched children who had no visual symptoms (Group C). It was found that well organized VEP waveforms over one or both hemispheres (Types U and S), or those with a characteristic negative shift (Type N) suggest favorable prognosis. Integrated voltage of the VEP correlated well with long-term prognosis for visual recovery. The vertex VEP also (had) provided some predictions for visual prognosis. Overall results indicate good prognosis if related to sufficient voltage and complexity of the VEP components.

Purification and characterization of a new lipase from Fusarium sp. YM-30.

The extracellular lipase from Fusarium sp. YM-30 was purified by a procedure involving ultrafiltration, ammonium sulfate precipitation, and DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M column chromatographies. The purified lipase was homogeneous with 12kDa of molecular mass by SDS-PAGE, and had high specificities for mono- and diacylglycerols, but low toward triacylglycerols. The enzyme had maximum activity at pH 7.0 to 8.0 and 37 degrees C, and hydrolyzed digalactosyl diglyceride too.

Physical and physiological specification of magnetic pulse stimuli that produce cortical damage in rats.

The effects of transcranial magnetic pulse stimuli on the brain tissue of rats were examined. In Experiment I, 52 male albino rats received pulsed magnetic stimulation of the head. Stimulus intensity, number of stimulations, stimulated sites, and interval between last stimulation and sacrifice for neuropathological examination were varied. High stimulus intensity (2.8 T) and 100 or more stimulations produced clearly defined microvacuolar changes in the neuropil portion of cortical layers 2-6 (especially layers 3 and 4) in 12 of 24 animals. Fewer stimulations and lower intensities produced no such effects in 28 rats given that stimulation. Midline stimulation and stimulation over the left hemisphere produced similar results. No other brain, ocular, or spinal structures manifested such changes. Lesions were present in animals that had intervals up to 30 days between the last stimulation and perfusion. In Experiment II with 18 animals, compound motor action potentials (CMAPs) evoked by magnetic stimulation of the cortical motor area and recorded from the right lower extremity were examined. The electromyographic threshold was 0.83 T. Further increases in stimulus intensity produced increases in CMAP amplitude, up to approximately 1.9 T. It was noted that the lesion-producing intensity (2.8 T) was 0.9 T greater than the intensity needed for near-asymptotic reactions and was 3.4 times the CMAP threshold value.

Memory for conditioned taste aversions is diminished by transcranial magnetic stimulation.

Rats were allowed to drink distinctively flavored water and later received an IP injection of LiCl. In Phase I, between drinking and the onset of the mild malaise, Experimental Group rats received stimulations of the head and Controls received equivalent stimulations of the back. Later, when the flavor was again presented. Experimentals drank 10-15% more, indicating that they had forgotten to some extent that the flavor was associated with illness. In Phase II, the procedure was repeated with a different distinctive flavor. Again, the Experimentals drank more on the test day. In Phase III, all rats received stimulations of the back between tasting and illness. Experimentals and Controls were not different on the test day, indicating that 100 prior head stimulations did not interfere with the establishment of a new taste aversion. Histological examinations revealed no gross morphopathology. We conclude that 50 brief pulse transcranial magnetic stimulations may cause a retrograde memory disruption, but we find no evidence for an anterograde memory effect or for structural changes.

Stability in waking-sleep states in neonates as a predictor of long-term neurologic outcome.

Thirty-three full-term neonates were ranked blindly on a scale ranging from the least to the highest "risk" for future neurologic complications on the basis of available perinatal biographies, tests, and examinations performed during the newborn period. Four prolonged polygraphic-behavioral recordings were obtained one week apart beginning at ten days after birth. Five waking and sleep states were scored in each session as percentages of total observation time, giving a total of 20 scores for each baby to be subjected to analysis of variance. These measures also provided individual profile consistency or variability in maintaining waking-sleep states over the selected period of postnatal time. The whole cohort, except three infants who could not be followed adequately, was then reexamined periodically over a period ranging from 3 to 4 years (+/- 6 months) for neurologic and developmental assessments. Except for two scores that produced a low level of statistical significance (P less than .05), the other 18 scores were found to be not associated with long-term outcomes. Even the first two scores were not satisfactory discriminators for the outcome of the individual babies. However, when coefficients of concordance (W) were computed from each individual baby profile, significant statistics (P less than .001) emerged to indicate good correlations between high or low W values in the newborn period and long-term outcomes. All 17 newborns who had W scores greater than 0.9 were found to be normal at follow-up regardless of the poor ranking given several of them during the newborn period. Among the 13 newborns who had W scores less than 0.9, 11 had a poor clinical outcome at follow-up, though several had been ranked initially as falling within the least "risk" group.

Neurophysiological, psychological, and nutritional investigations during discontinuation of the phenylalanine-restricted diet in children with classic phenylketonuria.

Assessment of cortical sensory and psychological functions in children with classic phenylketonuria during the time period of diet discontinuation did not uncover any significant changes when pre-diet and post-diet termination data were compared. Analysis of relationships among the study variables found that children who had adequate dietary control and who were of average or above average intelligence displayed normal somatosensory evoked potentials (SEP) whereas those children with less adequate dietary control and lower IQ scores had atypical SEP waveforms.

Wave form difference in visual evoked responses between normal and reading disabled children.

Wave form differences were found in visual evoked response between normal and reading disabled children. These differences were maximally seen over the left parietal area but were minimal at the vertex and over the occipital area.

The scalp topography of human somatosensory and auditory evoked potentials.

The waveform and topography of components of the scalp recorded somatosensory evoked poal (AEP) to click stimulation of the right ear, were determined for scalp electrode locations of the 10-20 system and for locations at the eye, mastoids, and posterior neck. Twenty-one SEP and twenty-two AEP components were analyzed. Differentiation of neurogenic and myogenic components was attempted on the basis of localization and variability. Some components of extracranial origin, apparently originating in frontal musculature, were small in most experienced subjects and large in most experimentally naive subjects. These and other presumptive myogenic potentials can distort adjacent neurogenic components. These data should aid in predicting SEP and AEP characteristics and in assessing myogenic distortion of neurogenic components.

Auditory evoked response: meaningfulness of stimuli and interhemispheric asymmetry.

Interhemispheric asymmetries of different magnitudes were observed in human cortical auditory evoked responses to speech and sound-effect stimuli. The wave with peak asymmetry occurred 100 milliseconds after signal onset. The amount of asymmetry of the amplitude of this wave was related to the meaningfulness to the subject of the auditory stimulus rather than to the mere use of verbal versus nonverbal materials.