The Effects of Adjuvants on Autoimmune Responses Against Testicular Antigens in Mice

Experimental autoimmune orchitis (EAO) is a model of immunologic male infertility and pathologically characterized by lymphocytic inflammation, which causes breakdown of the testicular immune privilege with spermatogenic disturbance. Generally, murine EAO is induced by immunization with testicular homogenate (TH) from the testes of donor mice + complete Freund''s adjuvant (CFA) + Bordetella pertussigens (BP), and it has been considered that treatment with these two adjuvants is required to enhance the immune response against testicular antigens. However, there remains a possibility that CFA and BP may affect autoimmune responses against the testicular antigens without TH. In the present study, we examined this possibility using real-time RT-PCR, Western blotting and immunohistochemical staining. The results demonstrated that immunization with TH in combination with CFA and BP evoked more severe EAO than that with only TH. Real-time RT-PCR analyses revealed that Fas mRNA expression in TH+CFA+BP-induced EAO was significantly higher than that in TH-induced EAO. Interestingly, IL-6 mRNA expression dramatically increased in TH+CFA+BP-induced EAO; however, no apparent change in IL-6 mRNA expression occurred in TH-induced EAO. It was also noted that treatment with CFA and BP alone augmented autoimmune reactions against some testicular autoantigens. These results indicates that these adjuvants are helpful in evoking severe EAO, and treatment with the adjuvants alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no TH.     

Nucleotide sequence of myostatin gene and its developmental expression in skeletal muscles of Japanese Black cattle

Myostatin, a member of the transforming growth factor‐β superfamily, is a well known negative regulator of skeletal muscle growth. In the present study, the 6660 bp nucleotide sequence of the myostatin gene in Japanese Black cattle (JBC), including the entire coding region of 1128 bp, was determined. The amino acid sequence deduced from the nucleotide sequence of JBC was well conserved with its sequence of other cattle, although it was found that an Α→G transition at nucleotide position 641 results in the substitution of asparagine by serine at amino acid position 214. In order to examine the expression pattern of the myostatin gene in the skeletal muscles of JBC, its expression in three skeletal muscles, Semitendinosus (ST) muscle, Biceps femoris muscle and Longissimus lumborum muscle, of fetal and calf stages was analyzed by real time polymerase chain reaction. The highest level of the myostatin expression was observed in the fetal stage. In calf stages the highest expression was observed in ST muscle compared with the other two muscles. These results suggest that a higher expression of myostatin gene, especially in the fetal stage and in ST muscle during calf stages, is involved in the arrest in skeletal muscle growth and that its functional domains and genomic structure in JBC are well conserved with those in other mammals.     

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-VPR’ lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar ‘06-LA’ and a deep-colored cultivar ‘Spectrum’ produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the ‘Spectrum’ parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.     

Population structure of pigs determined by single nucleotide polymorphisms observed in assembled expressed sequence tags

We have collected more than 190 000 porcine expressed sequence tags (ESTs) from full‐length complementary DNA (cDNA) libraries and identified more than 2800 single nucleotide polymorphisms (SNPs). In this study, we tentatively chose 222 SNPs observed in assembled ESTs to study pigs of different breeds; 104 were selected by comparing the cDNA sequences of a Meishan pig and samples of three‐way cross pigs (Landrace, Large White, and Duroc: LWD), and 118 were selected from LWD samples. To evaluate the genetic variation between the chosen SNPs from pig breeds, we determined the genotypes for 192 pig samples (11 pig groups) from our DNA reference panel with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Of the 222 reference SNPs, 186 were successfully genotyped. A neighbor‐joining tree showed that the pig groups were classified into two large clusters, namely, Euro‐American and East Asian pig populations. F‐statistics and the analysis of molecular variance of Euro‐American pig groups revealed that approximately 25% of the genetic variations occurred because of intergroup differences. As the F_IS values were less than the F_ST values_, the clustering, based on the Bayesian inference, implied that there was strong genetic differentiation among pig groups and less divergence within the groups in our samples.     

Prevalence of Giardia intestinalis infection in household cats of Tohoku district in Japan

Fecal samples obtained from 600 household cats (244 males and 356 females) kept in 3 prefectures of Tohoku district in Japan were examined for Giardia intestinalis antigen, using a commercial enzyme-linked immunosorbent assay (ELISA) kit. G. intestinalis antigen was detected in 40% of the fecal specimens. The factors such as the age, life style or environmental condition of cats could be significantly related to the positive rate of G. intestinalis antigen. In contrast, the investigative district, appearance of feces, sex, breed or origin produced no significant difference in the positive rate. The present results suggest that G. intestinalis infection is widely spread in household cats of Tohoku district.     

Genetic and biological characteristics ofTyphula ishikariensis isolates from Norway

Isolates ofTyphula ishikariensis, a snow mold fungus, were collected from five localities in Norway. They were divided into three groups according to genetics, cultural morphology, etc. Group I grew normally at 10 °C. Its mating patterns with Japanese taxa were variable: compatible with both biotypes A and B; compatible with biotype A but incompatible with biotype B; and incompatible with both biotypes. Group I was prevalent in southern inland districts such as Buskerud, Oppland, and Hedmark. Group II had smaller sclerotia as compared to other groups, and its sclerotia were often covered with white mycelium in nature and in culture. Group II was compatible with biotype B only. Group III was characterized by irregular growth at 10 ° C and genetic incompatibility with biotypes A and B. Cultural morphology of group III resembled that of group I at 0 °C. Rind cell patterns of sclerotia did not separate these two groups or biotypes. Isolates of groups II and III were often obtained from coastal regions in Finmark. Mating reactions were variable: monokaryons were compatible with their respective dikaryons, and monokaryons of groups II and III occasionally mated with group I dikaryons. Dikaryons of groups II and III, however did dikaryotize monokaryons of other groups. Norwegian isolates ofT. ishikariensis were highly variable, and the orthodox nomenclature system seemed inapplicalbe at the infraspecific level.