Identification and expression analysis of cDNA encoding insulin-like growth factor 2 in horses.

Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5'- and 3'-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses.

PCR detection of Babesia ovata from cattle reared in Japan and clinical significance of coinfection with Theileria orientalis.

We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis.

Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice.

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. Here we show that a spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA.

Molecular survey for Anaplasma bovis and Anaplasma phagocytophilum infection in cattle in a pastureland where sika deer appear in Hokkaido, Japan.

Anaplasma bovis and Anaplasma phagocytophilum infection were examined by species-specific nested polymerase chain reaction in cattle in a pastureland where sika deer appear in Hokkaido, Japan. Of the 78 cattle examined, 12 (15%) and 1 (1%) tested positive for infection by A. bovis and A. phagocytophilum, respectively. One cattle tested positive for both. A. phagocytophilum infection rates were significantly lower in cattle than in sika deer (46%), but the infection rate by A. bovis was not significantly different between cattle (15%) and sika deer (23%). The strain of A. phagocytophilum detected in this study may possess significantly lower virulence or infectivity in cattle hosts. No clinical symptoms were recorded in the positive cattle, and morulae were not detected in the blood smears.

Epidemiological survey of Theileria orientalis infection in grazing cattle in the eastern part of Hokkaido, Japan.

Theileria orientalis is one of the benign species of Theileria that is widely distributed in Japan and is sometimes responsible for serious economic losses in the livestock industry. In the present study, we surveyed the current status of T. orientalis infection in grazing cattle in the eastern areas of Hokkaido (Taiki, Otofuke, Shintoku, and Shin-Hidaka districts) using molecular methods, as well as traditional methods, of diagnosis. The genes encoding the major piroplasm surface protein (MPSP) and p23 of T. orientalis were identified using highly detectable polymerase chain reaction (PCR). Results of the MPSP-PCR assay indicated that grazing cattle in these districts, after about 1.5 months pasturage, showed high rates of infection, ranging from 10.0-64.8%. Although the main MPSP and p23 genotypes detected were the Ikeda- or Chitose-types, an MPSP gene closely relating to that found in Okinawa prefecture, and a p23 gene closely relating to the Australian (Warwick) Buffeli-type gene, were found in the cattle in Shintoku and Shin-Hidaka districts. The present survey indicated that there were at least five types of T. orientalis classified by their MPSP genes in Hokkaido, Japan, and that T. orientalis infection rates are still high in this region.

Distinct contribution of IL-6, TNF-alpha, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice.

Cytokines play key roles in spontaneous CD4(+) T cell-mediated chronic autoimmune arthritis in SKG mice, a new model of rheumatoid arthritis. Genetic deficiency in IL-6 completely suppressed the development of arthritis in SKG mice, irrespective of the persistence of circulating rheumatoid factor. Either IL-1 or TNF-alpha deficiency retarded the onset of arthritis and substantially reduced its incidence and severity. IL-10 deficiency, on the other hand, exacerbated disease, whereas IL-4 or IFN-gamma deficiency did not alter the disease course. Synovial fluid of arthritic SKG mice contained high amounts of IL-6, TNF-alpha, and IL-1, in accord with active transcription of these cytokine genes in the afflicted joints. Notably, immunohistochemistry revealed that distinct subsets of synovial cells produced different cytokines in the inflamed synovium: the superficial synovial lining cells mainly produced IL-1 and TNF-alpha, whereas scattered subsynovial cells produced IL-6. Thus, IL-6, IL-1, TNF-alpha, and IL-10 play distinct roles in the development of SKG arthritis; arthritogenic CD4(+) T cells are not required to skew to either Th1 or Th2; and the appearance of rheumatoid factor is independent of joint inflammation. The results also indicate that targeting not only each cytokine but also each cell population secreting distinct cytokines could be an effective treatment of rheumatoid arthritis.

Synthesis of pyrrolidine-fused [34]- and [36]octaphyrins via 1,3-dipolar cycloaddition.

[reaction: see text] The utility of expanded porphyrins as a dipolarophile in cycloaddition reactions has been investigated. The 1,3-dipolar cycloaddition of meso-octakis(pentafluorophenyl)[36]octaphyrin(1.1.1.1.1.1.1.1) with an azomethine ylide provides mono- and bis-pyrrolidine-fused octaphyrins regio- and stereoselectively. Treatment of the cycloadduct with MnO(2) afforded [34]octaphyrin quantitatively.

Plasma and urine levels of electrolytes, urea and steroid hormones involved in osmoregulation of cetaceans.

Cetaceans are well adapted to their hyperosmotic environment by properly developed osmoregulatory ability. A question here is how they regulate water and mineral balances in marine habitats. In the present study, we determined blood and urine levels of various chemicals involved in osmoregulation, compared them with those in artiodactyls, and characterized the values in the whales. Blood and urine samples obtained from baleen whales of common minke (Balaenoptera acutorostrata), sei (B. borealis), and Bryde's whales (B. brydei), and toothed whales of sperm whales (Physeter macrocephalus) were analyzed for osmolality, major electrolytes, urea, steroid hormones and glucose. The urine osmolality and Na(+) concentrations in the cetaceans were much higher than those in the cattle. Furthermore, the cetaceans had 5 to 11-fold urea in plasma than the cattle, and 2 to 4-fold urea in urine. There were no significant difference in the plasma concentrations of corticosteroids between the cetaceans and the cattle. The present results indicate that the osmoregulatory parameters seem to be not affected by the reproductive stage and sex steroid hormones. The concentrations of urea in plasma and urine of the baleen whales were higher than those of the sperm whales, indicating a possibility that their osmoregulatory mechanisms may be correlated to their feeding habits. The present results suggest that cetaceans have unique osmoregulatory mechanisms by which they excrete strongly hypertonic urine to maintain fluid homeostasis in marine habitats.

Nitrogen digestion and urea recycling in Hokkaido native horses fed hay-based diets.

Nitrogen (N) digestion and urea-N metabolism in Hokkaido native horses fed roughage-based diets containing different types and levels of protein sources were studied. Horses (173 ± 4.8 kg) fitted with an ileum cannula were fed four diets consisting of 100% timothy hay (TH), 88% TH and 12% soybean meal (SBM), 79% TH and 21% SBM, and 51% TH and 49% alfalfa hay at 2.2% of body weight. Dietary protein content varied from 5% to 15% of dry matter. Apparent N digestibilities in the pre-cecum and total tract for the TH diet were lower than those for other diets. However, the proportion of post-ileum N digestion to N intake was not affected by the diets. Urea-N production was linearly related to N intake, but gut urea-N entry was not affected by the diets. The proportion of gut urea-N entry to urea-N production tended to be higher for the TH diet (57%) than the two SBM diets (39%). Anabolic use of urea-N entering the gut was not affected by the diets (20-36% of gut urea-N entry). These results indicate that urea-N recycling provides additional N sources for microbial fermentation in the hindgut of Hokkaido native horses fed low-quality roughages.

Low-molecular-weight dextran infusion is more effective for the treatment of hemoconcentration due to severe ovarian hyperstimulation syndrome than human albumin infusion.

The most severe complication of ovarian hyperstimulation syndrome (OHSS) is thromboembolism, which is related to hemoconcentration. Dextran 40 infusion has greater effectiveness for the treatment of hemoconcentration due to OHSS than does human albumin infusion.

Use of a rigid-type polyethylene T-cannula, silicone tubing, and adhesion netting for surgical cannulation of ileum of the horse.

A rigid-type of polyethylene T-cannula was fitted into the anterior ileum of six horses in order to improve the cannulation techniques. A piece of polyethylene net was fastened onto the intestinal wall around the cannula to prevent dislodgment of the cannula by promoting a secure adhesion between the ileum and the abdominal wall. The cannula barrel sheathed with silicone tubing was exteriorized through a stab incision at the lateral ventral wall on the transverse line of the second lumber vertebra, and a flange was screwed onto the barrel. The feeding regime gradually increased concentrate without roughage prevented any colic signs. The use of these techniques succeeded in the ileal cannulation with no leakage of digesta.

Surgical repair of a pseudoaneurysm derived from a nine-year-old saphenous vein graft after coronary artery bypass.

A 57-year-old male presented intermittent pain between the left shoulder and neck. He had undergone coronary artery bypass twice and all grafts had been confirmed to be angiographically intact at three and a half years prior. Coronary angiography revealed that a pseudoaneurysm derived from around the proximal ascending aortic anastomosis of the saphenous vein graft that connected to the left anterior descending branch and #4-atrioventricular branch sequentially. The pseudoaneurysm compressed the saphenous vein graft itself. He underwent repair of the pseudoaneurysm through median sternotomy after left subclavian artery-saphenous vein graft bypass utilizing a free left radial artery without cardiopulmonary bypass through left anterolateral thoracotomy. A 7 mm length longitudinal tear in the saphenous vein graft near the proximal anastomosis had caused the pseudoaneurysm. This tear had likely been caused by perforation by a catheter during coronary arteriography three and a half years ago.

Genetic diversity of Theileria orientalis in tick vectors detected in Hokkaido and Okinawa, Japan.

In the present study, we investigated the possible tick vectors that can transmit Theileria orientalis in eastern Hokkaido, Japan. Questing ticks collected from three different districts, Taiki, Otofuke, and Shin-Hidaka, of Hokkaido included Ixodes persulcatus, Haemaphysalis megaspinosa, Haemaphysalis douglasi, and Ixodes ovatus, while all the ticks collected from Yonaguni island of Okinawa were identified as Haemaphysalis longicornis. When the ticks were screened by polymerase chain reaction (PCR) for T. orientalis, the parasite was commonly detected among all tick species. Genotype-specific PCR assays revealed that all tick species in Hokkaido were predominantly detected with type 2, while ticks collected from Okinawa (H. longicornis) were predominantly detected with type 1. Consistent with the genetic diversity of T. orientalis in ticks, genotyping PCR assays from cattle grazed in the same Hokkaido sampling locations identified type 2 as the most prevalent genotype. This study provides the first identification of I. persulcatus, H. megaspinosa, H. douglasi, and I. ovatus as possible tick vectors of T. orientalis, and finds that the variety of vectors apparently capable of transmitting T. orientalis is wider in Japan than expected. The authors suggest that tick control strategies should be modified in Hokkaido based on the seasonal activities of ticks identified in the present study.

Differences in grazing behavior of horses and cattle at the feeding station scale on woodland pasture.

In this study, grazing behavior of mares and cows was compared on woodland pastures, in the context of foraging hierarchy. A horse-group (12 native Hokkaido horses) and a cattle-group (5 Hereford cows) were grazed on the woodland pastures of 13.3 ha and 5.8 ha, respectively. They grazed mainly on Sasa nipponica. Grazing behavior at a feeding station (FS) scale was recorded for three focal animals in each group. Mares took one bite per FS at a high percentage of FSs (24.3%) and cows took one, two and three bites per FS at high percentage (20.3%, 19.2% and 15.4%, respectively). Although feeding patch (FP) scale for both animals could be defined as a group of FSs, the length of FP and grazing behavior at FP scale were similar for both animals. The number of steps taken between two adjacent FSs or FPs for mares was larger than that for cows. The results suggested that both horses and cattle had similar foraging hierarchy from bite to FP scale but grazing behavior of horses at the FS scale was different from that of cattle, suggesting that grazing behavior of large herbivores has greater similarity in larger foraging hierarchies.

Regioselective borylation of porphyrins by C-H bond activation under iridium catalysis to afford useful building blocks for porphyrin assemblies.

Highly regioselective and efficient borylation of a variety of porphyrins has been achieved by reaction with bis(pinacolato)diboron through C-H bond activation under iridium catalysis on the basis of the synthetic protocol developed by Miyaura, Hartwig, and Smith. A boryl group can be selectively introduced at sterically uncongested positions in the peripheral aryl groups of porphyrin substrates whose peripheral beta-positions are sterically hindered. Curiously, beta substituents adjacent to the aryl group to be borylated have unexpectedly large effects on the regioselectivity, because the iridium catalyst can discriminate between subtle steric differences. Chemoselective borylation was also achieved for several functionalized porphyrins. This borylation protocol can be applied to various monomeric and oligomeric functional porphyrins, hence offering an efficient route to elaborate multiporphyrin-based molecular constructs.

Spontaneous development of autoimmune arthritis due to genetic anomaly of T cell signal transduction: Part 1.

A point mutation of the gene encoding ZAP-70, a key signal transduction molecule in T cells, results in spontaneous development of T cell-mediated autoimmune arthritis in mice homozygous for the mutation. The genetic anomaly alters differentiation and selection of T cells in the thymus, leading to thymic production of arthritogenic autoimmune T cells. The arthritogenic T cells persist in the periphery and elicit arthritis when activated by microbial agents that stimulate innate immunity. This model is instrumental in understanding how genetic variations in T cell signal transduction, together with environmental influences, contribute to the development of autoimmune disease.

Highly regioselective Ir-catalyzed beta-borylation of porphyrins via C-H bond activation and construction of beta-beta-linked diporphyrin.

Highly regioselective borylation of 5,15-diaryl- and 5,10,15-triarylporphyrins has been achieved by the reaction with bis(pinacolato)diborane under iridium catalysis. Borylated porphyrins are a useful building block for constructing porphyrin arrays, hence offering an effective route to beta-functionalized porphyrins.