Increased lipid oxidation heralds diabetes onset in DR.lyp/lyp rats.

AIM: The BB rat model of type 1 diabetes exhibits altered body weight gain and body temperature regulation prior to hyperglycemia onset, implying the existence of as yet unidentified biomarkers of autoimmune processes that destroy pancreatic beta cells. To investigate this hypothesis, we compared the metabolic profile of diabetes-resistant DR.lyp/+ rats and their diabetes-prone, congenic DR.lyp/lyp littermates in the days leading up to diabetes onset. METHODS: Except for the Gimap5 mutation on chromosome 4, congenic DR.lyp/lyp rats are genetically identical to DR.lyp/+ littermates. They invariably develop hyperglycemia at 46-81 days of age, whereas DR.lyp/+ rats do not develop diabetes. In addition to daily food intake and body weight, indirect calorimetry was performed continuously on male DR.lyp/lyp and DR.lyp/+ rats (n=6/group) for 6-18 days to measure locomotor activity, VO (2), VCO (2) and RQ. RESULTS: DR.lyp/lyp rats exhibited a progressive decrease of RQ compared to DR.lyp/+ rats 0.005+/-0.001 units/day (p<0.005). Limiting the analysis to the six days prior to diabetes onset revealed a larger decrease of 0.007+/-0.002 units/day (p<0.001) in DR.lyp/lyp animals, whereas RQ of the DR.lyp/+ rats remained unchanged. This metabolic change occurred prior to hyperglycemia onset and was not associated with changes of any other parameter. CONCLUSIONS: Diabetes onset in DR.lyp/lyp rats is heralded by a progressive shift towards lipid oxidation relative to carbohydrate metabolism.

Reaction of 7-(2-mesyloxy-2-phenylethyl)theophylline with amines: Synthesis of 1,2,3,6-tetrahydro-6-imino-2-oxo-7H-purine derivatives.

Theophylline was converted to 7-(2-phenyl-2-methanesulfonyloxy)ethyl congener and the product was treated with ammonia or primary amines in a mixture solution of water and organic solvents. Two products were proven to be the styrene analogue and 7-(2-amino-2-phenylethyl)theophylline. The structure of the third product was elucidated as the 1,2,3,6-tetrahydro-6-imino-2-oxo-7H-purine derivatives by spectroscopic analysis including HMBC correlation and X-ray crystallography.

Monoclonal antibodies inhibiting invasion of cultured cells by Eimeria tenella sporozoites.

Monoclonal antibodies (MoAbs) secreted by 12 hybridomas and reactive with the surface antigens of E. tenella sporozoites were produced. These MoAbs designated as KC-1 to KC-12 were characterized as IgG3 kappa. In Western blot analysis, these MoAbs reacted with only one polypeptide band of sporozoite antigens having a molecular mass of 25 kDa. All the MoAbs were reactive with sporozoites, trophozoites, immature and mature first generation schizonts, first generation merozoites, and the interior structure of sporulating oocysts of E. tenella, however, not with any of the methanol-fixed sporozoites of E. acervulina, E. mitis, E. maxima, E. brunetti, E. necatrix and E. praecox. Invasion of primary culture of chicken kidney cells by E. tenella sporozoites was significantly inhibited by several of the MoAbs. These results suggest that these MoAbs recognizing the E. tenella-specific surface molecule are involved in the invasion of sporozoites into host cells.

Influence of calcium concentration on the antimicrobial activity of lasalocid against Selenomonas ruminantium.

The present study was designed to examine the interaction between the effects of lasalocid and Ca2+ on the growth and structure of Selenomonas ruminantium HD-4. Lasalocid, at a dose of 10 microM, inhibited cell growth almost completely after 12 hr incubation in the presence of relatively high extracellular concentrations of Ca2+ (from 5 to 50 mM), but only slightly reduced cell growth in the presence of 0.2 mM Ca2+. With Ca2+ alone, cell growth was also inhibited at 12 hr as a function of the concentration of Ca2+ over the range 5 to 50 mM. In cultures at mid-exponential phase, growth was markedly inhibited by the simultaneous addition of 10 microM lasalocid and 15 mM Ca2+, but only moderately inhibited by lasalocid in the absence of Ca2+. However, there was no significant effect on bacterial growth at the mid-exponential phase when Ca2+ alone was added to the incubation medium. In thin sections of cells treated with lasalocid in the presence of 15 mM Ca2+, abnormal cells were found with cytoplasmic voids and with an outer membrane detached from the inner membrane layer, this change in the outer membrane was also found in cells treated with lasalocid alone. There was no visible abnormality in the outer membrane in thin sections of Ca2(+)-treated cells, while most of these cells showed only a slight contraction of cytoplasmic material or a loss of cytoplasmic contents. These results indicate the presence of a synergistic effect between the actions of lasalocid and Ca2+ on cell growth, presumably due to induced cytoplasmic alterations.

Intestinal ciliates found in the feces of Japanese native Tokara pony, with the description of a new genus and a new species.

The composition of intestinal ciliates excreted in the feces of 20 Tokara native ponies kept in Iriki farm of Kagoshima University was surveyed. Eleven genera consisting of 18 species were identified based on the description of Strelkow. One new genus including a new species, Wolskana tokarensis n. gen., n. sp., of the family Buetschliidae was recognized and described. The average ciliate density of all horses was 13.8 x 10(3)/ml. Those of stallions (n = 5), mares (n = 11) and foals (n = 4) were 3.9, 16.1 and 43.3 x 10(3)/ml respectively. The value of stallions was significantly lower and that of foals higher than that of mares. Brillouin's diversity index of all horses was 1.554 on average. The index of stallions, 1.470, was significantly lower than those of mares, 1.539, and foals, 1.698. The average number of species per host was 11.7.

Medication of feedlot calves infected with Eimeria spp. by a combination of sulfamonomethoxine and ormetoprim.

Feedlot calves naturally infected with Eimeria spp. were medicated by a combination of sulfamonomethoxine and ormetoprim (Ektecin). Calves, less than one year old and positive for coccidiosis, were administered with Ektecin (5, 10 and 20 ml/100 kg of body weight/day) and Daimeton (100% sulfamonomethoxine: 5 g/100 kg of body weight/day) for five days. No diarrhea were found on and after 3 days of medication in all the groups, and no oocysts were detected on and after day 5, 2, 1 or 3 from calves of the respective medicated groups. In samples from a group of calves administered with lowest dose of Ektecin, eimerian oocysts of 4 species were detected on day 0, and additionally 3 species (totally 7 species) were found on day 3 of medication.

Antimicrobial activity of lasalocid against Selenomonas ruminantium--effect of changes in pH induced by changing glucose concentration.

A significant decrease in pH occurred in the culture medium when cells of Selenomonas ruminantium HD-4 were incubated in the presence of relatively high concentrations of glucose (0.4 and 1.0%). Forty microM lasalocid reduced cell growth to 35.5 and 35.7% of control growth, respectively, for 0.05 and 0.4% glucose, while growth was completely inhibited by 40 microM lasalocid in the presence of 1.0% glucose. In the presence of 80 microM lasalocid, cells were unable to grow within 24 hr at any glucose concentration. In the case of treatment with 10 microM lasalocid, growth was inhibited when pH decreased below 6.0 in the presence of 0.4 and 1.0% glucose. With 0.05% glucose, pH never reduced to below 6.4 within 24 hr and there was no significant effect of lasalocid on either growth or pH. When cultures were treated with 10 microM lasalocid at the mid-exponential phase, growth was moderately inhibited and abnormal cells which had outer membrane detached from the inner membrane layer resulting in cell lysis were found in thin sections. These results indicate that the presence of large amounts of glucose decreases pH in culture medium and that the decrease in pH to below about 6.0 causes the increased effect of lasalocid on the growth of S. ruminantium. These results suggest that the antimicrobial activity of lasalocid is potentiated by a decrease in extracellular pH.

Biochemical and antigenical characterization of tannin-protein complex degrading enterobacteria isolated from koalas, Phascolarctos cinereus.

Biochemical and antigenical characteristics of tannin-protein complex degrading enterobacteria (T-PCDE) isolated from Koalas, Phascolarctos cinereus, were investigated. T-PCDE had a specific profile of characteristics, and T-PCDE was distinguished from those of 12 type strains of Enterobacteriaceae used.

Identification of a new bovine MHC class II DRB allele by nucleotide sequencing and an analysis of phylogenetic relationships.

Three overlapping cDNA clones coding for the bovine major histocompatibility complex (MHC) class II DR beta chain were isolated. A clone NR1 encoded a primary translated product of 266 amino acids, 29 of which were deduced to form a signal peptide and 237 to form the mature polypeptide. The protein predicted from this cDNA appeared to have all the features expected of an expressed MHC class II molecule. Comparison of the sequences and construction of a phylogenetic tree revealed that NR1 represents a BoLA-DRB3 gene and not a BoLA-DRB1 or BoLA-DRB2 pseudogene. NR1 and ovine sequences exhibited the greatest overall similarity among sequences from various mammalian species, followed by the equivalent human sequences. Indeed, the bovine allele was more closely related to certain ovine alleles than to other bovine alleles. A large number of replacement substitutions were identified when beta 1 domains encoded by NR1 and each of the 36 distinct BoLA-DRB3 alleles were compared, and most of the allelic variations were found in regions that are commonly polymorphic in DRB sequences from different species and correspond to the predicted antigen-recognition site. Thus, the predicted structure of the unique NR1 allele for BoLA-DRB3 further confirms the overall conservation of the product of this locus, as previously established from studies in rodent and man.

Rumen ciliates of Tokara native goat in Kagoshima, Japan.

The composition of rumen ciliate protozoa was surveyed in 15 Tokara native goats, Capra hircus domesticus, inhibiting Kagoshima, Japan. As a result, of 11 species with 3 forms under 5 genera detected, Entodinium simplex, E. nanellum and E. rectangulatum were found in all the animals examined. The large ophryoscolecid ciliates detected were Polyplastron multivesiculatum and Metadinium affine alone, which have been regarded as the typical members of Type A composition proposed by Eadie. Since Tokara native goats have been bred isolated in a small islet for a long time, native goats that had been kept in south Japan might possess Type A fauna. Average ciliate density was 43.9 x 10(4) per 1 ml of rumen fluid. The number of species per host was 8.3 on average. Diversity index was 1.412 on average.

Detection of serum antibody against bovine coccidiosis by using Eimeria tenella antigen.

Antigens of Eimeria tenella sporulated oocysts were applied to detect serum antibody of calf infected with Eimeria. Polypeptides of E. tenella oocyst were strongly proved with the infected bovine serum in immunoblotting. The infected serum also showed higher absorbances than that of normal serum in enzyme linked immunosorbent assay (ELISA) using E. tenella oocyst antigen. Suitable conditions for the ELISA were obtained by using soluble antigen of E. tenella oocysts at 2.5 micrograms/ml and bovine serum at 200 fold dilution.

Cross reactivity of serum antibodies from chickens immunized with three Eimerian species.

Cross reactivity of serum antibodies from chicken immunized with E. tenella, E. necatrix and E. acervulina was observed. The enzyme linked immunosorbent assay (ELISA) using soluble antigen prepared from oocysts of each species showed the cross reactivity among chicken serum immunized with the three species of protozoa, especially between E. tenella and E. necatrix. In immunoblot analysis with E. tenella oocyst antigen, 7 major bands were detected by homologous E. tenella antiserum and 10 and 6 bands were reacted by E. necatrix and E. acervulina antiserum respectively. In these antigens, the band of 33 kDa was regarded as cross reactive agent between E. tenella and E. necatrix because of high reactivity to both of the antiserum. In addition, the 130 kDa antigen that reacted with every antiserum was regarded as common antigen among these three species.

Antibacterial activity of tilmicosin against Pasteurella multocida and Actinobacillus pleuropneumoniae isolated from pneumonic lesions in swine.

Sixty one strains of Pasteurella multocida and 35 strains of Actinobacillus pleuropneumoniae isolated from pneumonic lesions of porcine lungs during the period from 1985 to 1989 in Japan were tested for antibiotic susceptibility to chlortetracycline (CTC), thiamphenicol (TP), tylosin (TS), acetylisovaleryl-tylosin (AIV-TS), tilmicosin (TMS), mirosamycin (MRM). Most strains of both species were sensitive to CTC, TP and TMS. Growth of fifty-one strains (83.6%) and forty-six strains (75.4%) of P. multocida were inhibited with 3.13 micrograms/ml of CTC and 0.78 micrograms/ml of TP, respectively. TS showed low activity against almost all strains (MIC > or = 6.25 micrograms/ml). Fifty-eight (95.1%), twenty-three (37.7%) and fifty (82%) of P. multocida showed MICs of > or = 6.25 micrograms/ml against AIV-TS, TMS and MRM, respectively. The MICs of A. pleuropneumoniae against CTC were less than 1.56 micrograms/ml. Thirty-two strains (91.4%) and 33 strains (94.3%) of A. pleuropneumoniae were inhibited with 3.13 micrograms/ml of TP and TMS respectively. However, TS, AIV-TS and MRM showed low activity against all of A. pleuropneumoniae (MIC > or = 6.25 micrograms/ml). Three different resistance patterns were observed in P. multocida and two in A. pleuropneumoniae isolates, respectively.

Cloning of cDNAs and the molecular evolution of a bovine MHC class II DRA gene.

Two overlapping cDNA clones coding for bovine major histocompatibility complex (MHC) class II antigen DRA chain were isolated and characterized. The full-length cDNA clone, MR1, encoded a primary translated product of 253 amino acids, 24 of which were deduced to be a signal peptide and 229 which formed a mature polypeptide. The amino acid sequences deduced from this clone resembled those of class II A molecules from other species in both size and structure, but no potential consensus site of N-linked glycosylation comparable to those in the human, mouse, rat and swine proteins was found in the alpha 2 domain, as well as ovine and equine DRA molecules. Comparison of amino acid sequences encoded by class II A genes among several species and a dendrogram constructed from these data places the DRA gene and the DQA/DYA genes on two distinct branches of a phylogenetic tree, with bovine DRA and ovine DRA being most similar on the DRA branch.

Rumen ciliate composition and diversity of Japanese beef black cattle in comparison with those of Holstein-Friesian cattle.

The composition of ciliate protozoa in the rumen of 30 Japanese beef black cattle (Bos taurus taurus) fed in Yoron Island, Kagoshima, was compared with that of 125 Holstein-Friesian cattle (Bos taurus taurus) fed in Hokkaido. Thirteen genera including 44 species with 32 formae detected from Japanese beef black cattle and 15 genera including 48 species with 25 formae from Holstein-Friesian cattle were identified. Thirty-six species with 19 formae were common to both hosts. A new species was recognized from Japanese beef black cattle and named Entodinium yunnense n. sp. This new species was classified into such four formae as yunnense n.f., spinonucleatum n.f., acutonucleatum n.f. and spinolobum n.f. A new forma of Entodinium bifidum having only one caudal spine was also recognized from Japanese beef black cattle and named monospinosum n.f. The average ciliate density in Japanese beef black cattle, 18.7 x 10(4)/ml, was lower than 40.3 x 10(4)/ml in Holstein-Friesian cattle. The average number of ciliate species per head in Japanese beef black cattle, 14.4 was also lower than 17.8 in Holstein-Friesian cattle. The average value of diversity index was 1.789 in Japanese beef black cattle and 1.718 in Holstein-Friesian cattle. Both incidence and composition ratio of Entodinium rectangulatum and E. ovinum were significantly higher in Japanese beef black cattle than in Holstein-Friesian cattle. On the other hand, Charonina ventriculi, E. caudatum, E. rostratum, E. parvum, E. bursa, Metadinium affine and Ostracodinium mammosum showed a reverse relation.

Antibiotic susceptibility of Mycoplasma hyopneumoniae isolated from swine.

The antibiotic susceptibility of thirty-nine strains of Mycoplasma hyopneumoniae isolated from swine between 1970-1981 and 1989-1990 was investigated. From the present results, it is suggested that the susceptibility to chlortetracycline has been decreasing in Japan. On the other hand, all the strains were sensitive to lincomycin, thiamphenicol and macrolides. Newly developed macrolides such as tilmicosin, acetyl-isovaleryl-tylosin and mirosamycin had equal or higher activity than general macrolides.

Susceptibility to Eimeria tenella of chickens and chicken embryos of partly inbred lines possessing homozygous major histocompatibility complex haplotypes.

Susceptibility to Eimeria tenella infection of chicken embryos and chickens of partly inbred lines possessing different major histocompatibility complex haplotypes was investigated. Chicken embryos of line H-B2 possessing B2 homozygous haplotype showed lower mortality and higher oocyst production than embryos of line H-B15 possessing B15 homozygous haplotype. Embryos of line H-B2 chickens were considered more resistant to E. tenella infection than those of line H-B15. Seven days after 10-day-old chickens were infected with E. tenella, the two lines showed no significant differences in percent bodyweight gain, cecal lesion score, and oocyst production; they differed significantly only in cecal shrinkage. Results suggest that the B system affects chicken embryos and chickens differently in susceptibility to E. tenella infection.

Enhanced release of an alveolar macrophage-derived chemoattractant for fibroblasts in rats after asbestos inhalation.

Our studies indicate the effects of in vivo asbestos exposure on the ability of alveolar macrophages (AM) to elaborate a chemoattractant for fibroblast using a rat model of asbestos inhalation. Two groups of rats were exposed by intermittent inhalation (6 hr/day for 5 days/week over a total period of 4 weeks) to either amphibole (crocidolite) or serpentine (chrysotile) asbestos. A group of control rats were sham-exposed to clean air only. The animals were sacrificed 2-5 months after the cessation of exposure. The AM were obtained from the 3 exposure groups in 2 different rat strains by the bronchoalveolar lavage and the cultured in RPMI-1640 medium for 24-96 hr at 37 degrees C. The supernatants from cultured AM were tested for chemotactic activity towards fetal rat skin fibroblasts in a chemotactic assay using 8 microns pore-size filters. The culture supernatants of AM obtained from crocidolite-exposed rats exhibited a significantly greater chemotactic activity towards rat fibroblasts than similar culture supernatants from sham-exposed control animals (p < 0.01) in both rat strains. Significant chemotactic activity was observed after chrysotile exposure (p < 0.05) in ACI rats but not in Fischer-344 rats. Maximal chemoattractant release from AM was noted after 48 hr in culture. Preliminary characterization of the chemoattractant has shown that it is a thermolabile and trypsin sensitive factor whose activity was partially reduced after dialysis. Since AM accumulate at sites of intrapulmonary asbestos deposition, these findings may have relevance to the pathologic accumulation of interstitial lung fibroblasts which occurs during asbestos-mediated lung injury.

Rumen ciliates of Ezo deer (Cervus nippon yesoensis) with the morphological comparison with those of cattle.

The composition of rumen ciliate protozoa was surveyed in 13 Ezo deer, Cervus nippon yesoensis, inhabiting Hokkaido, Japan. As a result, two species of the genus Entodinium, E. simplex and E. dubardi, were detected. Each Ezo deer had only a single species of protozoa, E. simplex or E. dubardi. Since E. simplex in Ezo deer had a wide variation in body size and shape, its measurements were compared with those of E. simplex, E. nanellum and E. exiguum obtained from Holstein-Friesian cattle feeding in the same area. The ciliate density ranged widely from 3.1 to 5882.4 x 10(3) with an average of 567.4 x 10(3) per 1 ml of rumen fluid.

The effects of asbestos inhalation on the distribution and enhancement of immunoassociated antigen expression of alveolar macrophage subpopulation.

We have studied the effects of in vivo asbestos exposure on the surface immune-associated (Ia) antigen expression and distribution of alveolar macrophage subpopulations defined by continuous iso-osmotic Percoll gradients (density range: 1.006 to 1.123 g/ml) using a rat model of asbestos inhalation. Two groups of rats were exposed by intermittent inhalation (6 hr/day for 5 days/week over 4 weeks) to either amphibole (crocidolite) or serpentine (chrysotile) asbestos. A group of control rats was sham-exposed to clean air only. Alveolar macrophages from rats of three groups were obtained by bronchoalveolar lavage. During exposure, distinct differences appeared within 7 days of asbestos exposure, and some of these findings persisted in the crocidolite-exposed group for as long as 2 to 5 months after the cessation of exposure. Furthermore, relatively greater proportions of Ia-antigen positive cells were detected in several density fractions obtained from both asbestos-exposed groups (especially the crocidolite-exposed group). Multinucleated alveolar macrophages were seen frequently in all Percoll fractions after both types of asbestos inhalation. A significant proportion of multinucleated alveolar macrophages in these fractions expressed surface Ia-antigen positivity. The finding of enriched numbers of higher-density phagocytes in bronchoalveolar lavage cell subpopulations from asbestos-exposed rats may reflect the presence of newly recruited-immature monocytes and/or macrophages at sites of intrapulmonary asbestos deposition. Also, increased proportions of Ia-antigen positive cells suggest that a part of them were functionally activated.