Detection of intra- and interspecific variation of the dry rot fungus <Emphasis Type="Italic">Serpula lacrymans</Emphasis> by PCR-RFLP and RAPD analysis

We investigated a genotype-based assay to discriminate the dry rot fungi Serpula lacrymans. DNAs were extracted from 74 isolates from the northern half of Japan, and internal transcribed spacers (ITS) were amplified by polymerase chain reaction. Genotypes of isolates were checked by restriction fragment length polymorphism (RFLP) using two enzymes, Taq I and Hha I. Among the 74 isolates identified as S. lacrymans in terms of morphologic features, 5 isolates were shown to have been misidentified. Random amplified polymorphic DNA (RAPD) analysis was conducted in order to detect the intraspecific diversity of S. lacrymans isolated in Japan. Because no relation between geographical origin and genetic distances was observed, the intraspecific diversity of S. lacrymans is suggested to be small.Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002     

Variation in chemical composition of corn dried distillers grains with solubles in relation to in situ protein degradation profiles in the rumen

Chemical composition and in situ degradation profiles were analyzed for 27 samples of dried distillers grains with solubles (DDGS) distributed in Japan, and a wide variation was found in neutral detergent fiber (NDF) content, which had positive relationships to detergent‐insoluble crude proteins such as neutral detergent‐insoluble crude protein (NDICP) and acid detergent‐insoluble CP (ADICP). Samples with lower NDF (< 35% on dry matter (DM)) showed higher soluble fractions of protein, but the degradation rate of microbially degradable protein in the rumen was not different in comparison with the samples with higher NDF, and no difference was shown between samples with higher and lower NDF after 24 and 48 h of in situ incubation for DM and CP degradation, respectively. The NDICP content in the digestion residue decreased with time of incubation, especially for samples with higher NDF, while the ADICP content increased. These results suggest that a part of the soluble fraction of CP in DDGS would be incorporated into NDICP by the heating process in bio‐ethanol production, which is still highly degradable, whereas another part of the fraction incorporated into ADICP would proceed to the advanced steps of irreversible amino‐carbonyl reaction.     

Evaluation of myelotoxicity in dietary restricted rats

The purpose of this study was to clarify the effect of decreased food consumption on evaluation of myelotoxicity in routine general toxicity studies. Male rats were divided into the following 7 groups: 12, 15, and 18 mg/kg 5-fluorouracil (5-FU) treatment groups (FU12, FU15 and FU18); dietary restriction groups (R12, R15 and R18 receiving the same amount of food as the rats in the FU12, FU15 and FU18 groups, respectively); and a nontreated control group (NT). We compared the changes in body weight, hematology and the results of cytological analyses of bone marrow and histopathology among the groups after administration and recovery periods of 14 and 7 days, respectively. At the end of the administration period, the FU15 and FU18 groups showed decreases in many hematologic and bone marrow parameters that were all similar to those in the corresponding dietary restriction groups (R15 and R18). A granulocyte abnormality (polyploidy: frequency of 1% or less) was also observed in all 5-FU treated groups. At the end of the recovery period, increases in the reticulocyte and platelet counts and extramedullary hematopoiesis of the spleen were observed in the 5-FU treated groups. These results indicate that the results of general toxicity studies in rats should be evaluated in consideration of dietary restriction effects when food consumption is decreased at about 30-40% or more. Careful morphological observation of hemocytes would be helpful in distinguishing the effect of a drug from that of dietary restriction in relation to hematological and bone marrow parameters. Performance of a recovery test to determine the reactive response of hematopoiesis is also recommended.     

Brazilian isolates of Trypanosoma (Megatrypanum) theileri: diagnosis and differentiation of isolates from cattle and water buffalo based on biological characteristics and randomly amplified DNA sequences

We detected and cultivated isolates of Trypanosoma (Megatrypanum) theileri from cattle and water buffaloes in S?o Paulo state, southeastern Brazil, which were characterized by comparing morphological, growth and molecular features. Although isolates from cattle and water buffalo were morphologically indistinguishable, they differed in their growth characteristics. A dendrogram based on randomly amplified polymorphic DNA (RAPD) patterns indicated close-genetic relationships among all isolates from both species, which were all tightly clustered together and distant from Megatrypanum species from wild mammals. In addition, isolates within the T. theileri-cluster were clearly segregated into two host-associated groups. The sequence of a synapomorphic RAPD-derived DNA fragment (Tth625), which was shared by all T. theileri trypanosomes from cattle and buffalo but not detected in any of 13 other trypanosome species, was used as target for a conventional T. theileri-specific PCR assay. We also defined RAPD fragments (Tthc606 and Tthb606) that distinguished cattle from buffalo isolates. Thus, distinct growth features and genetic variability distinguished between isolates from cattle and water buffaloes of the same geographic origin, suggesting an association of these isolates with their host species. The trypanosomes from water buffalo reported here are the first T. theileri-like isolates from the Asiatic buffalo (Bubalus bubalis) to be continuously cultured and compared with cattle isolates using biological and molecular methods.     

Pressure-flow relationship and longitudinal distribution of pulmonary vascular resistance in heartworm-infected dogs

The pressure-flow relationships and the longitudinal distributions of pulmonary vascular resistance in normal and heartworm-infected (HWI) dogs were compared in an isolated, blood perfused preparation. The pulmonary circulation was partitioned into pulmonary arterial, middle, and venous segment based on the concept of a five element lumped model. The pulmonary arterial pressure-flow relationships were found to be non-linear and convex to the pressure axis in both normal and HWI lungs. The pressure-flow relationships of the pulmonary arterial and venous segment were linear and these slopes in the HWI lungs were significantly higher than the normal lungs. The pressure gradient of the middle segment was increased as flow increased at lower flow range, however, it was not increased during higher perfusion range in both lungs. At higher flow, the pressure gradient of the middle segment in the HWI lungs was significantly higher than the normal lungs. These results suggest that the ohmic resistance was almost equal to the sum of the two slopes of the pressure-flow relationships of the pulmonary arterial and venous segment because the pressure gradient of the middle segment was not altered as flow increased during higher perfusion rate. Because the slopes of the pressure-flow relationships of the pulmonary arterial and venous segment were increased with heartworm infection, the ohmic resistance of HWI lungs would be higher than normal lungs. The intercept pressure on the pressure axis of the linear portion of the pulmonary arterial pressure-flow relationship, a critical closing pressure, was regarded as pressure gradient of the middle segment during higher perfusing rate because the intercept pressures of pressure-flow relationships of pulmonary arterial and venous segment were almost equal to zero. Therefore, the critical closing pressure of HWI lungs would be higher than normal lungs. The pulmonary hypertension of filariasis appears to be due to an increase in ohmic resistance and elevated critical closing pressure.     

Effects of fiber length and orientation on elasticity of fiber-reinforced plywood

Short carbon fibers, a reinforced material in wood veneer composites, were used to investigate the effects of fiber length and orientation of fibers on the elasticity of plywood. The technical feasibility, elasticity, and strength of the reinforced plywood with short carbon fiber were evaluated. In a short fiber reinforcement system, the fiber length does not directly influence the reinforcement in Cox's theory when the fiber length exceeded a certain length. When the length of short carbon fiber is beyond 3 mm, the high reinforced result was obtained in the experiment. However, if fiber length was too long, the reinforced result was less owing to the bridge between fibers and the increase of holes. The optimum fiber length must be considered. The orientation of fibers has a strong influence on the reinforcement. Unidirectional, perpendicular, and random orientation displayed different influence on the elasticity. Experimental results were discussed with Cox's method. Reinforced plywood with short carbon fibers in random orientation has a higher shear modulus and bending strength than the controls, in addition to other mechanical properties.     

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.     

Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein

Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzyme-linked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP(Sc)) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP(Sc) detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP(Sc) was observed in WB, even when performed after 4 days of incubation at 37 degrees C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP(Sc) due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses.     

Molecular basis of the shish-kebab morphology in polymer crystallization

In the rich and long-standing literature on the flow-induced formation of oriented precursors to polymer crystallization, it is often asserted that the longest, most extended chains are the dominant molecular species in the "shish" of the "shish-kebab" formation. We performed a critical examination of this widely held view, using deuterium labeling to distinguish different chain lengths within an overall distribution. Small-angle neutron-scattering patterns of the differently labeled materials showed that long chains are not overrepresented in the shish relative to their concentration in the material as a whole. We observed that the longest chains play a catalytic role, recruiting other chains adjacent to them into formation of the shish.