Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.     

Relationship between blood calcium level and ST peak interval of electrocardiographic variables in peripartum Holstein cows

The association between blood calcium levels and electrocardiographic variables was compared in 137 normal parturient and 36 peripartum recumbent Holstein cows to determine whether hypocalcemia in peripartum dairy cows can be rapidly diagnosed using electrocardiograph. Inverse of STc (ST peak interval/SS interval^0.5) and blood ionized calcium or serum calcium concentrations were strongly correlated, and both correlation coefficients were 0.81 (P<0.001). The 95% prediction interval indicated that cows with STc >0.385 ± 0.001 sec are very likely to be hypocalcemic (blood ionized or serum calcium concentrations of <0.9 mmol/l or <7.5 mg/dl, respectively). These findings indicate that hypocalcemia in parturient cows can be non-invasively estimated using the STc.     

A Recessive Lesion Mimic Mutant of Rice with Elevated Resistance to Fungal Pathogens

A gamma-ray-induced rice mutant, M1009, which spontaneously forms brown specks on leaves was isolated. In appearance, these lesions very much resemble those that are produced during the hypersensitive resistance response of rice to pathogens. A similar phenotype has been associated with a number of mutants in maize or Arabidopsis, which have been briefly categorized as disease lesion mimics. Brown speck formation was suppressed at temperatures of 25°C and above, and was severe at 20°C. Light irradiation is also required to initiate brown specks. In addition, the mutant exhibits heightened resistance to two major fungal pathogens of rice, Magnaporthe grisea and Cochliobolus miyabeanus. Genetic characterization of the mutant has indicated that its les-bs (lesion-brown speck) phenotype segregates as a monogenic recessive mutation. Received 13 September 1999/ Accepted in revised form 15 December 1999     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN) mice

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.     

Rumen ciliate protozoal fauna of reindeer in Inner Mongolia, China

Rumen ciliate species and composition were surveyed on the reindeer kept in Inner Mongolia, China. As a result of survey, all the reindeer had the same 18 species of 8 genera of the ciliates in their rumen. Appearance of Entodinium parvum is the first record in this host species. Of the species detected, 15 species including Entodinium anteronucleatum, E. bicornutum, Enoploplastron confluens and Epidinium gigas which have been detected only from reindeer were common with those in the reindeer kept in the other areas, such as Alaska, Russia and Finland, indicating that every rumen ciliate faunae in reindeer has high similarity to each other, and suggesting that these hosts have been isolated from the other ruminants for long time and established characteristic ciliate fauna. The ciliate density was estimated as 1.1-2.5 x 10 (6)/ml, which value was fairly higher than those in domestic ruminants. Surface structures of characteristic species were observed by SEM.     

Phenotypic variation and the relationships among mustard (Brassica juncea L.) germplasm from Pakistan

An investigation was conducted to determine the extent of diversity and relationships among the Brassica juncea germplasm from Pakistan using morphological characters. A total of 52 accessions, including the collected germplasm as well as commercial cultivars/improved lines, were studied under field conditions at Tsukuba, Japan during 1995 and 1996. All the accessions were characterized for 35 agro-morphological characters from seedling emergence to crop maturity. Morphological data were analyzed by numerical taxonomic techniques using two complementary procedures: cluster and principal component analyses. Phenograms based on Euclidean distance placed the accessions into six groups during both years. Landrace groups were primarily associated with morphological differences among the accessions and secondarily with the breeding objectives and horticultural uses. The mustard germplasm collected from Pakistan showed a comparatively low level of phenotypic variation amongst themselves and were genetically similar to the oilseed cultivars. However, the oilseed forms and vegetable cultivars were genetically distinct. This study revealed that the evaluated germplasm appears to have a narrow genetic base which undergoes a high level of genetic erosion. This is perhaps due to the use of the same ancestors in the selection of new lines for similar horticultural traits, replacement by major crops and changes in agricultural land uses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity in mustard (Brassica juncea L.) germplasm from Pakistan as determined by RAPDs

The genetic diversity and the relationships among a collection of mustard (B. juncea) germplasm, including 41 accessions collected from Pakistan, 6 oilseed cultivars/ lines and 5 Japanese vegetable cultivars, were evaluated using random amplified polymorphic DNA (RAPD) markers. A total of 198 polymorphic amplified products were obtained from 30 decamer primers. Of these, 14 were unique to the accession PAK-85835 and 37 were specific to PAK-85839. Based on pair-wise comparisons of RAPD amplification products, genetic similarity was estimated using similarity coefficients of Nei & Li (1979) and a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA). Cluster analysis based on these genetic similarities placed most of the collected germplasm and oilseed cultivars/lines close to each other, showing a low level of polymorphism between the oilseed accessions collected in Pakistan. However, the clusters formed by the oilseed collections and cultivars were distinct from those formed by the vegetable cultivars. A low level of genetic variability of oilseed mustard in Pakistan was attributed to the selection for similar traits and horticultural uses. The farmers' preference for more remunerative crops and perhaps the close parentage of these accessions further contributed towards their little diversity. The study demonstrated that the RAPD is a simple and fast technique to compare the genetic relationships and the patterns of variation among accessions of this crop. This revised version was published online in August 2006 with corrections to the Cover Date.