Immunological characteristics and response to lipopolysaccharide of mouse lines selectively bred with natural and acquired immunities

Genetic improvement of resistance to infectious diseases is a challenging goal in animal breeding. Infection resistance involves multiple immunological characteristics, including natural and acquired immunity. In the present study, we developed an experimental model based on genetic selection, to improve immunological phenotypes. We selectively established three mouse lines based on phagocytic activity, antibody production and the combination of these two phenotypes. We analyzed the immunological characteristics of these lines using a lipopolysaccharide (LPS), which is one of the main components of Gram‐negative bacteria. An intense immunological reaction was induced in each of the three mouse lines. Severe loss of body weight and liver damage were observed, and a high level of cytokine messenger RNA was detected in the liver tissue. The mouse line established using a combination of the two selection standards showed unique characteristics relative to the mouse lines selected on the basis of a single phenotype. Our results indicate that genetic selection and breeding is effective, even for immunological phenotypes with a relatively low heritability. Thus, it may be possible to improve resistance to infectious diseases by means of genetic selection.     

Spontaneous Basal cell carcinoma of the submandibular gland in a rat

At necropsy, a white nodule (about 5 × 3 mm in size) was observed in the right submandibular gland of a 10-week-old female GALAS rat. Histopathologically, oval to spindle-shaped and pale basophilic tumor cells proliferated closely, and formed variably sized foci. The nodule partially spread into or invaded the surrounding normal tissue, and necrotic foci were recognized in the tumor. Immunohistochemically, the nuclei of the tumor cells showed a diffusely positive reaction for p63, and the cytoplasm showed a diffusely positive reaction for cytokeratin and negative reaction for αSMA, vimentin, desmin and S-100. Many tumor cells were positive for PCNA. Ultrastructurally, the tumor cells contained many tonofilaments in the cytoplasm and a few desmosomes at the intercellular portion. Based on these findings, the tumor was diagnosed as a basal cell carcinoma originating from the duct in the rat submandibular gland.     

Detection of zearalenone and its metabolites in naturally contaminated porcine follicular fluid by using liquid chromatography-tandem mass spectrometry

Zearalenone (ZEN) and its metabolites are important nonsteroidal estrogenic mycotoxins that cause reproductive disorders in domestic animals, especially pigs. We aimed to simultaneously detect ZEN and its metabolites á-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in porcine follicular fluid (FF) by liquid chromatography-tandem mass spectrometry. ZEN and α-ZOL, but not β-ZOL, were detected in all pooled FF samples collected from coexisting follicles (diameter ≥ 6 mm) within 10 ovaries. Furthermore, ZEN and α-ZOL were detected in samples pretreated with β-glucuronidase/arylsulfatase, but not in those left untreated, suggesting that the FF samples contained glucuronide-conjugated forms of the mycotoxins that may be less harmful to porcine oocytes due to glucuronidation affecting the receptor binding. Nonetheless, the effects of the glucuronide-conjugated forms should be studied, both in vitro and in vivo.     

Effect of Escherichia coli wild type or its derivative with high nitrite reductase activity on in vitro ruminal methanogenesis and nitrate/nitrite reduction

The effects of two kinds of Escherichia coli strains, wild-type E. coli W3110 or E. coli nir-Ptac, which has enhanced nitrite reduction activity, on in vitro CH4 production and nitrate and nitrite reduction in cultures of mixed ruminal microorganisms was investigated using continuous incubation systems. Escherichia coli nir-Ptac, a derivative of wild-type E. coli W3110, was constructed by replacing self promoter of nir BD operon encoding subunits of nitrite reductase in E. coli W3110 by tac promoter to make the expression of nir BD higher and constitutive. The nitrite reductase activity of E. coli nir-Ptac was approximately twice as high as E. coli W3110. The culture media consisted of 400 mL of strained ruminal fluid taken from two nonlactating Holstein cows receiving a basal diet of orchardgrass hay at maintenance level (55 g of DM/kg of BW0.75 daily), and 400 mL of autoclaved artificial saliva. Treatments were arranged in two separate 3 x 3 factorials consisting of nitrate (NaNO3; 0, 5, or 10 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac, or nitrite (NaNO2; 0, 1 or 2 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac. The control culture contained no chemical or microbial additives. Escherichia coli cells were inoculated into in vitro mixed ruminal cultures at approximately 2 x 10(8) to 10(9) cells/mL. Methane production by ruminal microorganisms was decreased markedly (P < 0.001) by the addition of nitrate and nitrite, and by the inoculation of cultures with E. coli W3110 or E. coli nir-Ptac (P < 0.01). With mixed nitrite-containing cultures, E. coli nir-Ptac inhibited (P < 0.001) in vitro nitrite accumulation and CH4 production more than E. coli W3110, which may be due to the tac promoter-enhanced nitrite reductase activity of E. coli nir-Ptac accelerating electrons to be consumed for nitrite reduction rather than CH4 biosynthesis. In conclusion, anaerobic cultures of E. coli W3110 or E. coli nir-Ptac may decrease CH4 production in the rumen. The inoculation of E. coli W3110 or, especially, E. coli nir-Ptac to mixed ruminal microorganisms may decrease nitrite toxicity when ruminants consume high-nitrate-containing forages and when nitrite is applied to abate ruminal CH4 production.     

Cloning and characterization of the glutamate dehydrogenase gene in Streptococcus bovis

Streptococcus bovis, an etiologic agent of rumen acidosis in cattle, is a rumen bacterium that can grow in a chemically defined medium containing ammonia as a sole source of nitrogen. To understand its ability to assimilate inorganic ammonia, we focused on the function of glutamate dehydrogenase. In order to identify the gene encoding this enzyme, we first amplified an internal region of the gene by using degenerate primers corresponding to hexameric family I and NAD(P)⁺ binding motifs. Subsequently, inverse PCR was used to identify the whole gene, comprising an open reading frame of 1350 bp that encodes 449 amino acid residues that appear to have the substrate binding site of glutamate dehydrogenase observed in other organisms. Upon introduction of a recombinant plasmid harboring the gene into an Escherichia coli glutamate auxotroph lacking glutamate dehydrogenase and glutamate synthase, the transformants gained the ability to grow on minimal medium without glutamate supplementation. When cell extracts of the transformant were resolved by blue native polyacrylamide gel electrophoresis followed by activity staining, a single protein band appeared that corresponded to the size of S. bovis glutamate dehydrogenase. Based on these results, we concluded that the gene obtained encodes glutamate dehydrogenase in S. bovis.     

Institute of Animal Experimentation,Sapporo Medical University,Minami 1 Nishi 17, Sapporo 060, Japan

Asaccharolytic pigmented Porphyromonas species, including P. endodontalis, P. gingivalis, P. circumdentaria and unclassified species, were isolated from the plaque of adult dogs, but not from any oral sites of puppies and adolescent dogs. With age-dependency, the proportion of Porphyromonas species in the flora of plaque increased. Isolation of the genus Porphyromonas was clearly associated with the progress of periodontol disease. We suggested that Porphyromonas is the exogenous organism and obligate pathogen for canine periodontal diseases.     

Pacific Ocean and Japan Sea ecotypes of Japanese beech (Fagus crenata) differ in photosystem responses to continuous high light

Two ecotypes of Japanese beech (Fagus crenata Blume), the Pacific Ocean type (PAO) and the Japan Sea type (JAS), show different responses to high solar irradiance. When PAO and JAS saplings were grown in continuous high-light (H), leaves of JAS became pale green. To elucidate this phenomenon, we investigated in vivo photochemistry based on pigment concentrations of Photosystem (PS) I and PS II and Western blot analysis. In JAS-H leaves, the amount of D1-protein decreased, resulting in decreases in the maximal quantum yield of PS II (F(v)/F(m)) and electron transport rate, whereas PAO-H leaves maintained high activities. The PS I photochemistry determined by measurement of P-700 photo-oxidation showed that the intersystem electron pool size was 1.4 times greater in JAS-H leaves than in PAO-H leaves. Furthermore, the re-reduction kinetics of P-700(+) showed that cyclic electron transport around PS I was 1.2 times faster in PAO-H leaves than in JAS-H leaves. Analysis of the area over the fluorescence induction kinetics indicated that the relative abundance of the PS IIalpha center increased in PAO-H leaves, whereas JAS leaves were observed to have low acclimation capacity to high light. These results demonstrate that PAO leaves possess acclimation mechanisms to continuous high light, whereas JAS leaves are more vulnerable to continuous high light, resulting in reduced leaf longevity owing to photoinhibition caused by increases in the intersystem electron pool size and suppression of photochemistry at the level of PS I and PS II.     

Fabrication of structure-preserving monodisperse particles of PMMA-<Emphasis Type="Italic">grafted</Emphasis> fullerenes

Fullerene has fascinated characteristics and the fabrication of fullerene-containing assembly of high solubility in general solvents at facile way is immensely expected for the material processing. We aimed to control supramolecular structure of fullerene-containing molecules with arrested morphology, which would boost material potentials to wider application. In this study, we have synthesized poly(methyl methacrylate) (PMMA)-grafted fullerene and the association behavior of PMMA-grafted fullerene in solvent is utilized to obtain monodisperse assemblies with submicron size. Furthermore, we have succeeded to fix the assembly morphology by UV irradiation and recovered the assembly in another solvent in which the assembly basically does not form. This procedure enables us to obtain fullerene- dispersed materials at sub-μm order with various matrixes.