Search

A male Holstein-Friesian calf was born with multiple, cauliflower-like, pale pink cutaneous masses on the head and limbs. On histopathological examination, the cutaneous masses were diagnosed as congenital cutaneous fibropapillomatosis. Those lesions involved focal proliferation of sebaceous gland in the dermis. There were no histological findings to suggest bovine papillomavirus infection, such as the presence of intranuclear inclusion bodies, large keratohyalin granules, and koilocytosis. Furthermore, papillomaviral antigens and DNA were not detected by immunohistochemistry and polymerase chain reaction, respectively. These results suggested that there was no association between these cutaneous lesions and bovine papillomavirus infection, and the lesions were considered as harmartomatous changes.

Summary Histochemical organization of the Caudata olfactory system remains largely unknown, despite this amphibian order showing phylogenetic diversity in the development of the vomeronasal organ and its primary centre, the accessory olfactory bulb. Here, we investigated the glycoconjugate distribution in the olfactory bulb of a semi-aquatic salamander, the Japanese sword-tailed newt (Cynops ensicauda), by histochemical analysis of the lectins that were present. Eleven lectins showed a specific binding to the olfactory and vomeronasal nerves as well as to the olfactory glomeruli. Among them, succinylated wheat germ agglutinin (s-WGA), soya bean agglutinin (SBA), Bandeiraea simplicifolia lectin-I (BSL-I) and peanut agglutinin showed significantly different bindings to glomeruli between the main and accessory olfactory bulbs. We also found that s-WGA, SBA, BSL-I and Pisum sativum agglutinin preferentially bound to a rostral cluster of glomeruli in the main olfactory bulb. This finding suggests the presence of a functional subset of primary projections to the main olfactory system. Our results therefore demonstrated a region-specific glycoconjugate expression in the olfactory bulb of C. ensicauda, which would be related to a functional segregation of the olfactory system.

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4(+)CCR6(+)IL-7R(+)T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6(+)B helper T cells were phenotypically distinct from follicular helper T (T(FH)) cells and lacked BCL6 expression. In peripheral blood, a CCR6(+)T cell population with similar characteristics was identified, which lacked Th17- and T(FH)-associated gene signatures and differentiation-associated surface markers. CD4(+)CCR6(+)T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4(+)CCR6(+)IL-7R(+)T cells were associated with the presence of pathogenic anti-dsDNA (double-stranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10–producing CCR6(+)T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE.

Gene screenings have identified a number of reprogramming factors that induce pluripotency from somatic cells. However, the screening methods have mostly considered only factors that maintain pluripotency in embryonic stem cells, ignoring a potentially long list of other contributing factors involved. To expand the search, we developed a new screening method that examined 2,008 human genes in the generation of human induced pluripotent stem cells (iPSCs), including not only pluripotent genes but also differentiation-related genes that suppress pluripotency. We found the top 100 genes that increased reprogramming efficiency and discovered they contained many differentiation-related genes and homeobox genes. We selected two, HHEX and HLX, for further analysis. These genes enhanced the appearance of premature reprograming cells in the early phase of human iPSC induction, but had inhibitory effect on the late phase. In addition, when expressed in human iPSCs, HHEX and HLX interfered with the pluripotent state, indicating inverse effects on somatic reprograming and pluripotent maintenance. These results demonstrate that our screening is useful for identifying differentiation-related genes in somatic reprograming.

Physical exercise exerts favourable effects on brain health and quality of life of the elderly; some of these positive health effects are induced by the modulation of microbiota composition. We therefore conducted a randomised, double blind, placebo-controlled trial that assessed whether a combination of Bifidobacterium spp. supplementation and moderate resistance training improved the cognitive function and other health-related parameters in healthy elderly subjects. Over a 12-week period, 38 participants (66-78 years) underwent resistance training and were assigned to the probiotic Bifidobacterium supplementation (n=20; 1.25×1010 cfu each of Bifidobacterium longum subsp. longum BB536, B. longum subsp. infantis M-63, Bifidobacterium breve M-16V and B. breve B-3) or the placebo (n=18) group. At baseline and at 12 weeks, we assessed the cognitive function, using the Japanese version of the Montreal Cognitive Assessment instrument (MoCA-J); modified flanker task scores; depression-anxiety scores; body composition; and bowel habits. At 12 weeks, the MoCA-J scores showed a significant increase in both the groups, while the flanker task scores of the probiotic group increased more significantly than those of the placebo group (0.35±0.9 vs -0.29±1.1, P=0.056). Only the probiotic group showed a significant decrease in the depression-anxiety scores (5.2±6.3 to 3.4±5.5, P=0.012) and body mass index (24.0±2.8 to 23.5±2.8 kg/m2, P

This study was conducted to investigate the possibility of suppression of stress-induced cortisol (CORT) secretion by tryptophan (TRP) administration and to better understand its regulatory mechanisms by using a noradrenaline (NA) injection into the third ventricle (3V) as a stress model in cattle. A total of 25 Holstein steers with a cannula in the 3V were used. First, the increase in CORT secretion was observed following a NA injection into the 3V in a dose-dependent manner, verifying the appropriateness of this treatment as a stress model of CORT secretion (Experiment 1). The effect of prior-administration of TRP into peripheral blood with a dose that has been demonstrated to increase brain 5-hydroxytryptamine levels on the elevation of plasma CORT induced by NA or corticotropin-releasing hormone (CRH) was then examined (Experiment 2). The prior administration of TRP suppressed NA-induced, but not CRH-induced, CORT elevation. These results suggest that an increase in TRP absorption into peripheral blood could suppress the stress-induced CORT secretion in cattle via the attenuation of the stimulatory effect of NA on the hypothalamic CRH release.

Ghrelin is a multifunctional peptide that promotes an increase of food intake and stimulates GH secretion. Ghrelin secretion is regulated by nutritional status and nutrients. Although a high-protein (HP) diet increases plasma ghrelin secretion in mammals, the mechanisms and the roles of the elevated ghrelin concentrations due to a HP diet have not been fully established. To clarify the roles of elevated acylated ghrelin upon intake of a HP diet, we investigated the regulation of ghrelin concentrations in plasma and tissues in wethers fed with either the HP diet or the control (CNT) diet for 14 days, and examined the action of the elevated plasma ghrelin by using a ghrelin-receptor antagonist. The HP diet gradually increased the plasma acylated-ghrelin concentrations, but the CNT diet did not. Although the GH concentrations did not vary significantly across the groups, an injection of ghrelin-receptor antagonist enhanced insulin levels in circulation in the HP diet group. In the fundus region of the stomach, the ghrelin levels did not differ between the HP and CNT diet groups, whereas ghrelinO-acyltransferase mRNA levels were higher in the group fed with HP diet than those of the CNT diet group were. These results indicate that the HP diet elevated the plasma ghrelin levels by increasing its synthesis; this elevation strongly suppresses the appearance of insulin in the circulation of wethers, but it is not involved in GH secretion. Overall, our findings indicate a role of endogenous ghrelin action in secretion of insulin, which acts as a regulator after the consumption of a HP diet.

Background To resolve the shortage of donors associated with liver transplantation, the potential uncontrolled non-heart-beating donor (UNHBD) pool is expected to increase. However, warm ischemia-reperfusion injury leads to inferior survival in transplantation using the grafts from UNHBD compared with those from heart-beating donors. To overcome this problem, we developed a new method for preparation of liver grafts from UNHBDs consisting of a combination of short oxygenated warm perfusion (SOWP) and prostaglandin E1 (PGE1). Methods Using an ex vivo perfusion rat model, we examined the effectiveness of this new method. Results Using SOWP and PGE1 treatment, the total amount of bile production during reperfusion in UNHBD grafts was increased to the same level as that in the heart-beating donor grafts. The addition of PGE1 to SOWP buffer decreased aspartate aminotransferase/alanine aminotransferase and tumor necrosis factor α levels during 1 h of reperfusion. Necrosis and apoptosis were significantly decreased by SOWP + PGE1 treatment. SOWP + PGE1 ameliorated induction of mitochondrial permeability transition, and the total amount of mitochondrial cytochrome c in the SOWP + PGE1 group after reperfusion was kept significantly higher than that in the no treatment group. Cytosolic c-Jun N-terminal protein kinase activation was significantly suppressed by SOWP + PGE1. Decrease in mitochondrial Bcl-2 was suppressed by SOWP alone and SOWP + PGE1 treatment, and Bax in the mitochondria was significantly suppressed by SOWP + PGE1. Conclusion SOWP and PGE1 prior to cold preservation significantly improved the function of liver grafts that underwent warm ischemia-reperfusion injury. Therefore, this method might be useful in liver transplantation using UNHBD grafts.

The effects of cashew nut shell liquid (CNSL) feeding on methane production and rumen fermentation were investigated by repeatedly using 3 Holstein nonlactating cows with rumen fistulas. The cows were fed a concentrate and hay diet (6:4 ratio) for 4 wk (control period) followed by the same diet with a CNSL-containing pellet for the next 3 wk (CNSL period). Two trials were conducted using CNSL pellets blended with only silica (trial 1) or with several other ingredients (trial 2). Each pellet type was fed to cows to allow CNSL intake at 4 g/100 kg of body weight per day. Methane production was measured in a respiration chamber system, and energy balance, nutrient digestibility, and rumen microbial changes were monitored. Methane production per unit of dry matter intake decreased by 38.3 and 19.3% in CNSL feeding trials 1 and 2, respectively. Energy loss as methane emission decreased from 9.7 to 6.1% (trial 1) and from 8.4 to 7.0% (trial 2) with CNSL feeding, whereas the loss to feces (trial 1) and heat production (trial 2) increased. Retained energy did not differ between the control and CNSL periods. Digestibility of dry matter and gross energy decreased with CNSL feeding in trial 1, but did not differ in trial 2. Feeding CNSL caused a decrease in acetate and total short-chain fatty acid levels and an increase in propionate proportion in both trials. Relative copy number of methyl coenzyme-M reductase subunit A gene and its expression decreased with CNSL feeding. The relative abundance of fibrolytic or formate-producing species such as Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, and Treponema bryantii decreased, but species related to propionate production, including Prevotella ruminicolla, Selenomonas ruminantium, Anaerovibrio lipolytica, and Succinivibrio dextrinosolvens, increased. If used in a suitable formulation, CNSL acts as a potent methane-inhibiting and propionate-enhancing agent through the alteration of rumen microbiota without adversely affecting feed digestibility.

Hausp is a deubiquitinase that has been shown to regulate the p53–Mdm2 pathway. Cotransfection of p53 and Hausp stabilizes p53 through the removal of ubiquitin moieties from polyubiquitinated p53. Interestingly, knockout or RNA interference-mediated knockdown of Hausp in human cells also resulted in the stabilization of p53 due to the destabilization of Mdm2, suggesting a dynamic role of Hausp in p53 activation. To understand the physiological functions of Hausp, we generated hausp knockout mice. Hausp knockout mice die during early embryonic development between embryonic days E6.5 and E7.5. The hausp knockout embryos showed p53 activation, but no apparent increase in apoptosis. Embryonic lethality was caused by a dramatic reduction in proliferation and termination in development, in part due to p53 activation and/or abrogation of p53-independent functions. Although deletion of p53 did not completely rescue the embryonic lethality of the hausp knockout, embryonic development was extended in both hausp and p53 double knockout embryos. These data show that Hausp has a critical role in regulating the p53–Mdm2 pathway.

5'-Uridylic acid (UMP), which is present at high concentrations in cow's colostrum, has been shown to cause a reduction in increased plasma levels of insulin and glucose after ingestion of milk replacer in pre-weaning calves. However, the precise mechanisms of UMP action have not been investigated, and its action has not been investigated in other pre-weaning ruminants. In order to demonstrate whether UMP causes changes in postprandial metabolic and hormonal parameters in pre-weaning goats, 11 Saanen kids were given milk replacer (twice a day) without (n = 5) or with (n = 6) UMP (1 g for each meal, 2 g/day for each head) for 14 days. Analysis of blood samples taken in the morning of day 14 demonstrated that the feeding of milk replacer with UMP abolished the significant changes in postprandial plasma glucose, NEFA, GH and insulin concentrations induced by feeding of milk replacer alone, and demonstrated a tendency to increase IGF-I levels. However, there was no significant difference between the two groups at any sampling time. We conclude that UMP feeding with milk replacer showed a tendency to blunt the postprandial changes in levels of some plasma metabolites and hormones that are induced by replacer alone in pre-weaning goats.

GPR41 and 43 have recently been identified as G-protein-coupled cell-surface receptors for short-chain fatty acids (SCFAs). Bovine orthologs of GPR41 and 43 (bGPR41 and 43) mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and various lactation stages of bovine mammary gland. Acetate and propionate caused an increase in intracellular Ca(2+) concentrations in these cells that was blocked by the treatment with pertussis toxin (PTX). SCFAs significantly reduced forskolin-induced cAMP concentrations in these cells. The phosphorylation of mitogen-activated protein kinase (MAPK) p38 was selectively increased by SCFAs. The downstream substrate heat shock protein 27 (HSP27) was also phosphorylated by SCFAs at Ser-78 and -82, but not -15. These results suggest that bGPR41 mainly, but not bGPR43, mediate SCFA signaling in mammary epithelial cells and thereby plays some important role in mammary gland.

Although mammary epithelial cells are known to synthesize and accumulate triacylglycerol (TAG) in order to produce milk lipid in the cytosol, lipid and energy metabolism is still not fully understood. In this study, we assessed the effects of long-chain fatty acid (LCFA) on the accumulation of cytosolic TAG and uncoupling protein (UCP) 2 in cloned bovine mammary epithelial cells (bMEC). LCFAs significantly raised the expression of UCP2 mRNA and the accumulation of TAG. We observed the rapid elevation in UCP2 shown at 6h after LCFA treatment. Insulin (5-50 ng/ml) or dexamethasone (500 nM) significantly suppressed the expression of UCP2 mRNA. These results suggest that UCP2 play an important role of lipid and energy metabolism in mammary epithelial cells.

When duplicated sister chromatids are not properly compacted in mitosis, chromosomes are mis-segregated, inducing genetically unstable tetraploidy known to facilitate aneuploid malignancies. Here, we show that tetraploid cells produced by impaired chromosomal condensation are eliminated by a novel type of cell death different from caspase-dependent apoptosis. The cell death was associated with downregulation of eukaryotic translation elongation factor-1 alpha 1 (eEF1A1/EF-1alpha) expression in conjunction with accumulation of its mRNA in processing bodies (P bodies). Importantly, expression of exogenous eEF1A1 was shown to inhibit the caspase-independent cell death, and a similar cell death was observed after inducing the expression of short hairpin RNA specific for eEF1A1. Furthermore, the number of spontaneously arising binucleated cells was indicated to increase several fold during 1- to 2-week cultivation after initiation of exogenous eEF1A expression. Taken together, the novel cell death machinery should help to eliminate abnormal tetraploid cells and inhibit tumorigenesis.

Ghrelin and growth hormone (GH) play a key role in regulating energy balance, metabolic hormone secretion and food intake. Ghrelin and GH responses to dietary compositions have not yet been fully clarified, although there may be significant relationships between dietary compositions and ghrelin and GH responses. In the present study, therefore, we assessed whether dietary compositions influence postprandial plasma ghrelin and GH levels in wethers. Four wethers were respectively fed concentrate (C) or timothy hay (R) for 14 days. The levels of total digestive nutrients (TDN) and crude protein (CP) were adjusted to be at the same level. The basal ghrelin in both groups was rapidly and significantly decreased after feeding. Although the decline of ghrelin levels in C was greater and shorter than that in R, no significant difference was observed in the area under the curve (AUC) or in the incremental area. The plasma GH levels were also rapidly and significantly decreased after feeding in both groups and a significant difference was observed between the two groups for AUC of GH. Interestingly, the circadian changes in the plasma ghrelin levels were close to those in the GH levels in C, but this was not the case in R. These data suggest that dietary compositions influence postprandial plasma ghrelin and GH levels, and that these differences may be caused by several factors, including nutrients and ruminal fermentation.

Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.

The cellular target of curvularol, a G1-specific cell-cycle inhibitor of mammalian cells, was identified by a genetic approach in Saccharomyces cerevisiae. Since the wild-type W303 strain was highly resistant to curvularol, a drug hypersensitive parental strain was constructed in which various genes implicated in general drug resistance had been disrupted. Curvularol resistant mutants were isolated, and strains that exhibited a semi-dominant, curvularol-specific resistance phenotype were selected. All five strains examined were classified into a single genetic complementation group designated YCR1. A mutant gene responsible for curvularol resistance was identified as an allele of the RPL3 gene encoding the ribosomal protein L3. Sequence analysis of the mutant genes revealed that Trp255Cys and Trp255Leu substitutions of Rpl3p are responsible for curvularol resistance. Rpl3p mutants in which Trp255 residue was replaced by other amino acids were constructed. All of these replacements led to varying degrees of increased resistance to curvularol and growth defects.

We measured and compared plasma levels of GH and ghrelin in response to feeding in 4-week-old (milk replacer-fed) and 13-week-old (alfalfa hay cube-fed) goats, in order to elucidate whether or not the postprandial regulation of these hormone levels changes around weaning. Furthermore, we examined the effects of suckling from the dam or intravenous glucose administration on both hormone and insulin levels in kids. In 4-week-old goats, feeding of a milk replacer diet significantly increased plasma GH levels without changing level of ghrelin. In contrast, in 13-week-old goats, feeding of hay cubes did not change the levels of either ghrelin or GH. Suckling of milk directly from the dams significantly increased the levels of GH and insulin, but not ghrelin, in kids. Finally, intravenous injection of glucose (0.625 mmol/kg BW) did not cause any significant increase in the levels of GH or ghrelin, despite a significant increase in the levels of insulin and glucose. From these results, we conclude that the regulatory system of the somatotropic axis is altered by weaning or weaning-associated processes, and that ghrelin levels may not be involved in this alteration in young goats.

In order to assess the biological significance of weaning and water deprivation on the control of plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH) and metabolites in response to stimulation with arginine-vasopressin (AVP) and corticotropin-releasing hormone (CRH), we carried out three experiments in which male goats before and after weaning were intravenously injected with AVP or CRH alone, or in combination with each other. In experiment 1, 17-week-old (post-weaning) goats were intravenously injected with AVP or CRH alone at the doses of 0.1, 0.3 and 1.0 nmol/kg body weight (BW). The AVP injection significantly and dose dependently increased plasma levels of ACTH, cortisol, GH and metabolites, whereas the injection with CRH did not cause significant increases in the levels of these parameters. In experiment 2, 4-week-old (pre-weaning) and 13-week-old (post-weaning) goats were injected with either AVP or CRH alone, followed by a combined injection of both secretagogues at a dose of 0.3 nmol/kg BW. Although the basal levels of the hormones and metabolites, with the exception of glucose, were greater in the 4-week-old goats, the hormone responses induced by stimulation with AVP were weaker than those induced in 13-week-old goats. Additionally, there were no responses in any hormone patterns to CRH stimulation in 4-week-old goats. In experiment 3, 13-week-old goats were injected with CRH alone followed by injection with AVP for two consecutive days of water deprivation. The animals were subjected to withdrawal of up to 20% of the total blood volume and water deprivation for up to 28 h. However, no significant differences in plasma ACTH, cortisol or GH levels were observed between days 1 and 2. Based on these results, we concluded that: (1) AVP is a more potent stimulant than CRH in terms of its ability to induce increases in plasma levels of ACTH, cortisol and GH; (2) the role of AVP as a secretagogue of hypothalamus–pituitary–adrenal hormones is strengthened, whereas the ineffective role of CRH remains unaltered, by weaning; (3) acute stress such as massive withdrawal of blood volume and subjection to water deprivation may not be sufficient burdens to alter stress-related hormone levels in young goats.

Proinsulin C-peptide is involved in several biological activities. However, the role of C-peptide in vascular smooth muscle cells is unclear. We therefore investigated its effects, in vascular smooth muscle cells in high-glucose conditions.Rat aortic smooth muscle cells were cultured with 5.5 or 20 mmol/l glucose with or without C-peptide (1 to 100 nmol/l) for 3 weeks. Proliferation activities, the protein expression of platelet-derived growth factor (PDGF)-beta receptor, the phosphorylation of p42/p44 mitogen-activated protein (MAP) kinases, and glucose uptake were measured.The proliferation activities increased approximately three-fold under high-glucose conditions (p0.05). C-peptide suppressed hyperproliferation activities that were induced by high glucose. This happened in a dose-dependent manner from 1 to 100 nmol/l of C-peptide. C-peptide (10 and 100 nmol/l) inhibited the increased protein expression of PDGF-beta receptor and the phosphorylation of p42/p44 MAP kinases that had been induced by high glucose (p0.05). Furthermore, 100 nmol/l of C-peptide augmented the impaired glucose uptake in the high-glucose conditions.These observations suggest that C-peptide could prevent diabetic macroangiopathy by inhibiting smooth muscle cell growth and ameliorating glucose utilisation in smooth muscle cells. C-peptide may thus be a novel agent for treating diabetic macroangiopathy in patients with type 1 and type 2 diabetes.

Postprandial changes in plasma concentrations of GH, insulin, IGF-I, leptin and metabolites were compared between young Holstein bull calves fed with milk alone (control group) and with milk+5′-uridylic acid (UMP) (UMP group). UMP (2 g/day) was given with milk at 0830 h and 1530 h for 11 days from the 4th to the 14th day after birth. The perirenal fat weight was significantly lower in the UMP group than in the control group, but there was no significant difference in the weights of the liver, spleen and heart between the groups. Basal GH concentrations in the UMP group were slightly higher, but the postprandial increase in plasma insulin level and the area under the curve for insulin in the UMP group were significantly lower than those in the control group. There was no significant difference in IGF-I levels between the groups. In addition, the postprandial glucose concentrations were lower in the UMP group as reflected by the insulin level, and nonesterified fatty acid concentrations were not different. In the muscle (M. longissimus thoracis) sampled at 14 days of age, the triacylglycerol (TAG) content was significantly greater but glycogen content was significantly lower in the UMP group than in the control group. From these results, we have concluded that feeding 5′-UMP at 2 g/day for 11 days significantly alters TAG accumulation in the body and plasma concentrations of GH and insulin in young bull calves.

Changes in plasma concentrations of GH and insulin in response to feeding and stimulation with GH-releasing hormone (GHRH) or GH-releasing peptide (GHRP-6, a ligand for endogenous GH secretagogue receptors) were compared between 3-week-old (milk-fed) and 12-week-old (concentrate and hay-fed) calves. Feeding of a milk-replacer diet in 3-week-old animals significantly increased the basal (prefeeding) concentrations of GH, insulin and glucose in plasma, whereas feeding of concentrate and hay in 12-week-old animals did not cause a significant change in these traits. However, in the animals maintained on a milk-replacer diet until 12 weeks of age, postprandial plasma GH concentrations and AUC (area under the curve) were not different from those in the age-matched weaned group. The venous injection of either GHRH (0.25 μg/kg) or GHRP-6 (2.5 μg/kg) significantly increased plasma GH concentrations in both 3- and 12-week-old animals, but GH AUC was significantly greater in 3-week-old than in 12-week-old animals. Insulin concentration was transiently but significantly increased by the injection of GHRP-6 only in 12-week-old animals, the AUC being greater in 12-week-old than 3-week-old animals. From these results, we conclude that postprandial levels of plasma GH and insulin concentrations are altered after weaning and by aging, and that the quality of diets or development of the neuroendocrine functions in the digestive–pituitary system may be involved in this alteration.

SUMMARYNeonatal thymectomy induces autoimmune gastritis (AIG) in 40–70% of BALB/c mice. We presumed that induction of autoimmunity by polyinosinic:polycytidylic acid (poly I:C) might allow development of a more aggressive model of AIG. A group of BALB/c mice were thymectomized on day 3 after birth. Neonatal thymectomized mice were either injected with poly I:C or phosphate-buffered saline (PBS). Non thymectomized neonatal BALB/c mice were injected with only poly I:C. All neonatal thymectomized mice injected with poly I:C developed 3 cardinal features of AIG: (1) moderate to severe degree gastritis (2) presence of autoantibody to H+/K+ ATPase and (3) loss of parietal cells. However, only 70% of the PBS-treated neonatal thymectomized BALB/c mice developed some, but not, all features of AIG. A mild degree of AIG was seen in 12 of 31 nonthymectomized BALB/c mice administered with only poly I:C. Administration of poly I:C in neonatal thymectomized BALB/c mice in the first and second week appeared to be the most effective for induction of aggressive AIG. The levels of interleukin (IL)-6, IL-12p70, interferon-γ and tumour necrosis factor-α were significantly higher in poly I:C-injected thymectomized mice compared to PBS-injected neonatal thymectomized mice (P

The effects of acetate and butyrate on leptin and leptin receptor (OB-R) expression in bovine and rat anterior pituitary were examined. In bovine tissues, leptin gene expression using RT-PCR was observed in fat and anterior pituitary but not in liver. Isolated anterior pituitary cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) for 3 days were further cultured for 48 h in DMEM containing 10 mM acetate or butyrate or without any fatty acids as control. Western blot analysis revealed that the abundance of leptin protein was greater in the presence of acetate and butyrate than that for the control culture. Leptin abundance was increased in a dose- and time-dependent manner in bovine anterior pituitary cells. However, leptin expression in rat cells, of which the basal level was much greater than that in ovine cells, was significantly decreased by the culture with butyrate. In addition, we studied the effects of both fatty acids on OB-R mRNA expression using semi-quantitative RT-PCR. The results showed that butyrate significantly decreased the expression in both bovine and rat cells. These findings indicate that acetate and butyrate enhance leptin expression in bovine, but not in rat anterior pituitary cells while butyrate suppresses OB-Ra expression in both rat and bovine pituitaries.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums. Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc.

Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel β3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans., Endometriosis is a painful condition in which endometrial cells are found outside the womb. Here, the authors identify a peptide that specifically binds to a receptor expressed on endometrial epithelial cells and use it to induce apoptosis in both cultured cells and baboons with endometriosis.

This report deals with the pathology and genetic basis of dilated cardiomyopathy in 10 Holstein-Friesian cows aged 3-6 years, a disease similar to that reported in Simmental-Red Holstein and Holstein-Friesian cattle in several other countries. The main clinical signs were associated with systemic circulatory failure, and at necropsy the animals showed cardiomegaly, severe congestion and fibrosis of the liver, and systemic cardiac oedema. Histologically, hypertrophy and vacuolation of the cardiac muscle fibres and severe fibrosis were noted. Electron microscopically, the sarcoplasm of the hypertrophic fibres was seen to be filled with fine structures of low electron-density, together with thin filamentous material, suggesting myofibrillar lysis. The mitochondria showed increased size, an abnormal cristae pattern and vacuolation due to partial loss of cristae. Pedigree analysis of the affected cattle indicated an autosomal recessive mode of inheritance. The family line of this cardiomyopathy overlapped with that of hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle, the pathological aspects and inheritance mode of which were reported previously. The available evidence suggested a genetic association between these two pathologically distinct diseases.

The antibody response to coproantigens of Cryptosporidium parvum was examined in saliva and sera of calves experimentally infected with C. parvum . Coproantigens of C. parvum with approximate molecular masses of 17, 15 and less than 14 kDa were found in the feces of infected calves on day 3 or later, and 60 and 23 kDa coproantigens observed between days 4 and 9 post-infection, respectively. The antibody reactivity to the coproantigens was mainly attributable to IgA class antibodies in saliva and was detectable during the convalescent phase of infection. A 15 kDa protein isolated from the feces of infected calves by immunoaffinity adsorption using a monoclonal anti C. parvum antibody was recognized by IgA antibodies present in the saliva during the convalescent phase of infection. These results suggest that this coproantigen may be released from C. parvum sporozoites and may induce IgA antibody production in the mucosal immune system of infected calves.

Among 55 Butyrivibrio fibrisolvens strains five ribotypes of B. fibrisolvens were described on the basis of RFLP profiles of 16S rDNA regions obtained with restriction endonuclease HaeIII. In the phylogenetic tree, these ribotypes were located in the XIVa cluster of Gram-positive bacteria. Phenotypic differences of selected ribotype groups became the basis for further reclassification of B. fibrisolvens.

Replacement of external NaCl with LiCl induced cytoplasmic alkalinization in CCL-39 cells and rat L6 myoblasts expressing the endogenous Na+/H+ exchanger isoform NHE1. This Li+-induced alkalinization is due to activation of the Na+/H+ exchanger because it was completely inhibited by 100 microM ethylisopropylamiloride (apparent Kd=1 microM) and because it did not occur in exchanger-deficient PS120 cells. The effect of Li+ was not mimicked by Na+, K+, Cs+ and choline+. Li+ caused cytoplasmic alkalinization in PS120 cells expressing NHE1 or NHE2, but not NHE3, when Li+ was added to cells at a concentration high enough to saturate their external transport sites as predicted from Li+ affinities. Li+ did not induce phosphatidylinositol (PI) turnover or intracellular Ca2+ mobilization. Li+-induced alkalinization was not affected by protein kinase C down-regulation, loss of glycogen synthase kinase 3beta caused by antisense oligonucleotide treatment, or pretreatment with calphostin C, pertussis toxin, MEK inhibitor PD98059 and PI3-kinase inhibitor LY294002. However, it was markedly suppressed by the tyrosine kinase inhibitor genistein (10 microM). Thus, externally added Li+ activates NHE1 and NHE2 via a mechanism possibly involving a tyrosine kinase, causing an increase in cytoplasmic pH that could potentially affect various cell functions.

Thrombin is a multifunctional serine protease. It is generated in inflammatory processes and induces the proliferation and chemotaxis of a variety of cells including mesothelial cells (MTCs). MTCs are epithelial cells derived from the mesoderm, as are the vascular endothelial cells. Since thrombin acts on endothelial cells to produce platelet-activating factor (PAF) and endothelin (ET)-1, it was hypothesized that MTCs also produce PAF and ET via the action of thrombin. Rat pleural MTC (RMTC, 4/4 R.M.-4) monolayers were cultural in tissue culture dishes for various periods. The supernatants were fractionated by means of high-performance liquid chromatography to determine the ET isoforms and PAF species present. Immunoreactive ET was measured using an enzyme-linked immunosorbent assay, and PAF was measured by means of a bioassay using a platelet aggregometer. ET-1, ET-2 and ET-3 were detected in RMTC-conditioned medium, and the predominant isoforms were ET-1 and ET-2. RMTCs mainly released C16:0 PAF into the supernatant. Immunoreactive ET and PAF were released via the action of thrombin. Synthetic PAF significantly induced secretion of ET, but the PAF receptor antagonists, WEB2086 and E6123, failed to modulate thrombin-induced ET release. These results indicate that thrombin acts on pleural rat mesothelial cells to release ET and PAF, which may play a role in the development of pleurisy.

We have previously reported that oxygenated warm perfusion prior to cold preservation (preperfusion) improved the function and viability of liver grafts from non-heart-beating donors (NHBD) using an ex vivo perfusion model. In this study, we evaluated the signaling pathway underlying these effects as well as the additive effect of preperfusion administration of edaravone, a free radical scavenger. Preperfusion treatment suppressed activation of JNK, p38 MAPK, and ERK. The addition of edaravone provided an insignificant increase in bile production and a trend to a decrease in TUNEL-positive cells. Oxygenated perfusion prior to cold preservation improved the function and viability of the grafts from NHBD, which accompanied impairment of MAPK activation. Moreover, the addition of edaravone significantly enhanced the effects of preperfusion.

Mutations within the growth hormone receptor (GHR) gene that lead to an inactivated or truncated GHR protein cause abnormal growth and small adult size in a variety of species (Laron dwarfism). We studied a line of miniature Bos indicus cattle that have phenotypic (small mature size) and endocrine (increased blood growth hormone and decreased blood insulin-like growth factor-I concentrations) similarities to Laron dwarfs. Liver mRNA from miniature and control cattle was used to amplify a cDNA within the coding region of the GHR. The miniature cattle had GHR mRNA size (determined by Northern blot) and cDNA sequence that were similar to control cattle and, therefore, were unlike most Laron dwarf genotypes in which the GHR gene is mutated. Amounts of mRNA from liver as well as muscle (superficial neck and longissimus) were analyzed by ribonuclease protection assay for IGF-I, total GHR, GHR 1A (inducible, liver-specific GHR mRNA), and GHR 1B (constitutive GHR mRNA). Four control and five miniature bulls were tested. As expected, liver IGF-I mRNA was decreased in the miniature cattle (approximately 12% of control; P < 0.01). The amount of the total GHR as well as GHR 1A mRNA were also decreased in liver (17% and 19% of control, respectively; P < 0.01). Other GHR mRNA, including GHR 1B mRNA, were similar for miniature and control cattle. In muscle, there was a tendency (P < 0.10) for decreased IGF-I mRNA and increased GHR mRNA in miniature compared with control cattle. In summary, a novel phenotype for Laron dwarfism in Bos indicus cattle was associated with underexpression of GHR 1A mRNA, but not other GHR mRNA variants in liver. In addition to decreased GHR 1A mRNA, the miniature cattle had decreased liver IGF-I mRNA. Full expression of GHR 1A in liver, therefore, may be required for full liver IGF-I expression and normal growth.

Objective and Design: To investigate whether mast cells (MCs) and chymase, the major protease of murine MCs, were involved in a chronic fibroproliferative disorder of the paws associated with type II collagen (CII)-induced arthritis.¶Materials: Eighteen DBA/1J mice were divided into 3 groups and were used to study fibroproliferative changes in paws elicited by immunization.¶Treatment: Arthritis was induced by immunization with CII, which was intradermally injected as an emulsion made with adjuvant. A booster shot was done 3 weeks after the initial shot. A group with no treatment and that received adjuvant alone served as control.¶Methods: Twelve weeks after the booster shot, inflammation of the paws was evaluated for pathological and biochemical indices. Chymase activity was determined with a chromogenic peptide substrate.¶Results: In CII-immunized group, collagen bundles accumulated around the destructed joints. In accordance with the pathological findings, MC density in the affected paws was increased (154.8 ± 13.3/mm2; p < 0.05 vs. control) and chymase activity was also increased (29.5 ± 2.8 mU/mg protein; p < 0.01 vs. control).¶Conclusions: The present results demonstrate increases in MCs and chymase in fibroproliferative paws of collagen-induced arthritic mice.

The effects of KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a novel Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure (ARF) in rats were examined. ARF was induced by clamping the left renal pedicle for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was markedly diminished in ARF rats. Pretreatment with KB-R7943 (10 mg/kg, i.v.) markedly attenuated the ARF-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damage, which was suppressed by KB-R7943. Activation of the reverse mode of Na+/Ca2+ exchange seems to play an important role in the pathogenesis of ARF. A selective Na+/Ca2+ exchange inhibitor may be useful in cases of ARF.

Because it is generally believed that apoptosis is not associated with inflammation, we hypothesized that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation. However, we recently found that the interaction led to the production of proinflammatory cytokines but not antiinflammatory cytokines, although the apoptotic cell membranes appeared to be intact. In this study, we examined in detail the relationship among the kinetics of apoptosis, phagocytosis and production of cytokines by macrophages. Among the time points examined, murine CTLL-2 cells became apoptotic in terms of cell size and exposure of phosphatidylserine after 12 h of culture in the absence of IL-2, and at the same time they began to be phagocytosed and lead to proinflammatory cytokine production by PMA-treated THP-1 cells (human macrophages). The phagocytosis of apoptotic cells by macrophages was also confirmed by confocal laser microscopy. The coculturing of human macrophages with murine apoptotic cells led to the production of human proinflammatory cytokines, notably IL-8, at both the mRNA level and the protein level. The coculturing of monocyte-derived macrophages with the apoptotic cells also led to the production of IL-8 protein. Both the phagocytosis and production of the cytokines were suppressed by either phospho-l-serine or RGDS (Arg-Gly-Asp-Ser), but not by RGES (Arg-Gly-Glu-Ser). Thus, the production of proinflammatory cytokines and phagocytosis of apoptotic CTLL-2 cells appear to be closely interrelated.

Our objective was to correlate hormonal changes with the timing and onset of estrus in heifers before and after luteolysis was induced with PGF2 alpha at two stages of the estrous cycle: d 6 to 9 (early; n = 10) or d 14 to 15 (late; n = 10). Blood was collected at intervals of 2 or 12 h to quantify serum concentrations of progesterone, estradiol-17 beta, and LH while heifers were observed visually for estrus and monitored for standing activity by pressure-sensitive, radiotelemetric devices. Although the concentrations of estradiol-17 beta that were associated with the putative appearance of the first dominant follicle declined before luteolysis was induced early in the cycle, some heifers that were given PGF2 alpha were in estrus as early as 35 h. Compared with heifers treated late in the estrous cycle, heifers that were treated early in the cycle produced less progesterone before PGF2 alpha treatment and had greater peak concentrations of estradiol-17 beta at estrus. In addition, heifers that were treated early in the cycle had shorter intervals from PGF2 alpha treatment to estrus, to peak estradiol-17 beta, and to peak LH and to initiation of estrus after the peak in estradiol-17 beta than did heifers treated later in the cycle. The increase in estradiol-17 beta associated with the putative first-wave follicle of the subsequent cycle and the duration of that cycle in early cycle heifers was less than after late cycle luteolysis. Results indicated that greater concentrations of estradiol-17 beta during estrus may be related to the durations of previous cycles and less progesterone exposure before luteolysis. The onset of estrus corresponded closely to, but preceded, the preovulatory LH surge by approximately 3 h.

Background. Tissue factor (TF) is a membranous protein normally present on the surface of the fibroblasts and smooth muscle cells of vessels. TF is an initiation factor for blood coagulation, and its expression is induced on macrophages and endothelial cells during the inflammatory or immune response. We studied the significance of TF expression in warm ischemic-reperfusion injury of the liver using a rat model. Methods. Following laparotomy of Lewis rats, the branches of the hepatic artery and portal vein leading to the median, left, and caudate lobes of the liver were clamped for 2 hr. The liver was reperfused after 120 min of ischemia. Rats were killed at 0, 1, 3, 5, 8, and 12 hr after reperfusion, and liver tissues were harvested. TF activity was measured by the chromophilic substrate S-2222. TF expression was studied by immunohistochemical staining with the monoclonal antibody HTF-K108. Results. TF activity in the blood showed a peak at 3 hr after reperfusion (8.960.5 U/L), then decreased and returned to the normal level by 12 hr (0.960.3 U/L). TF activity in ischemic liver tissue increased gradually over 12 hr after reperfusion (12236275 U/g dry weight before ischemia and 25456284 U/g weight at 12 hr after reperfusion). Histologically spotty necroses were observed in the liver tissue 5 hr after reperfusion. The necrotic area extended and encompassed almost all of the ischemic liver by 12 hr after reperfusion. Histochemically, TF staining was negative on the hepatocytes and slightly positive on sinusoid cells of the normal liver. On the other hand, TF was strongly stained, especially on the hypertrophic monocytic cells accumulating at the site of the necrosis, but staining was not evident on the necrotic hepatocytes. A slight degree of TF staining was observed on the alveolar epithelium of the lung, irrespective of liver ischemia and reperfusion. Conclusion. These results demonstrate that TF plays an important role in the development of the hepatic ischemic-reperfusion injury, and the subsequent microcirculatory incompetence might cause the formation of microthrombus and the development of necrosis. Ischemic reperfusion injury of the liver is a serious clinical problem in hepatic failure, hepatectomy, and liver transplantation. Recent studies of ischemia and reperfusion of the liver have focused on the function of neutrophils, the action mechanism of inflammatory cytokines, and oxygen free radicals

An Eubacterium ruminantium xylanase gene (xynA) was inserted into pYK4, a shuttle vector replicable in both Escherichia coli and Butyrivibrio fibrisolvens, and the resultant chimeric plasmid (pYK4XT) was electroporated into B. fibrisolvens OB156C in an attempt to obtain a more xylanolytic B. fibrisolvens. Electrotransformants were screened by the development of erythromycin resistance, followed by an activity staining and Southern hybridization. The presence of mRNA from xynA in the transformant, B. fibrisolvens NO4, was confirmed by Northern hybridization. Xylanase activity of the transformant NO4 was apparently enhanced regardless of carbon sources in the medium. When grown on glucose or cellobiose. NO4 had approximately 5-6 times higher intracellular activity than the parent OB156C on a culture volume basis as well as protein basis. The transformant showed extracellular xylanase activity much higher (between 7- and 10(4)-fold) than the parent. Transformant NO4 recorded the highest activity when grown on xylan. Most (90%) of the activity was extracellular. The extracellular activity was 2-fold greater in NO4. These findings indicate that the introduced xynA was expressed constitutively and the xylanase protein was exported into the culture supernatant. Growth of NO4 on glucose was similar to that of OB156C, which suggests little extra load for plasmid maintenance and foreign xylanase production in the transformant. The plasmid pYK4XT was maintained stably in the transformant for more than 100 generations.

Differences in trabecular and cortical bone loss have been demonstrated clinically, but differences in bone loss at different skeletal sites remain unclear. We examined regional variations in bone loss histomorphometrically in two strains of mice in which osteopenia progresses spontaneously: tiptoe-walking Yoshimura (twy) mice (from 4 to 37 weeks of age) and senescent ICR mice (from 4 to 88 weeks of age). Morphometrical measurements were obtained to investigate the changes with age in trabecular bone area and anterior cortical bone width in the lumbar vertebral body, trabecular bone area in the tibia, bone area in the parietal bone, and the cortical index in the humerus. Results showed that, in twy mice, trabecular turnover was higher than in ICR mice, and bone loss progressed in the following order: tibial trabecular bone, lumbar trabecular bone, parietal bone, lumbar anterior cortical bone, and the humerus. In ICR mice, bone formation declined after 60 weeks. Bone loss progressed in tibial trabecular bone and the parietal bone at 60 weeks of age, followed by lumbar trabecular bone, lumbar anterior cortical bone, and the humerus at 88 weeks of age. Bone loss varied at each site and between the two mouse strains, with different bone turnover rates. The findings of the present study indicate that special attention should be paid to regional variations in the progression of bone loss associated with differences in pathologic features.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine repeat within the androgen receptor (AR). We have studied the mutant AR in an in vitro system, and find both aggregation and proteolytic processing of the AR protein to occur in a polyglutamine repeat length-dependent manner. In addition, we find the aberrant metabolism of expanded repeat AR to be coupled to cellular toxicity, indicating a likely molecular basis for the toxic gain of AR function that produces neuronal degeneration in SBMA.

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes.

Organic cation transporters (OCTs) are expressed mainly in the kidney and liver. OCTs transport intrinsic organic cations, including monoamine, dopamine, serotonine and choline, across the plasma membrane. Here, we demonstrate that OCT2 (SLC22A2) is expressed in cholinergic neurons, motoneurons in the anterior horn of the spinal cord, and is implicated in acetylcholine (Ach) recycling in presynaptic terminals. Application of rabbit anti-peptide antibody revealed that OCT2 was expressed in the anterior horn of the spinal cord. Double immunostaining of muscle sections with anti-OCT2 and alpha-bungarotoxin (BTX) revealed that OCT2 was localized in the neuromuscular junctions (NMJs). Immunoelectron microscopy revealed that OCT2 was localized both in synaptic vesicles (SVs) in presynaptic terminals around the motoneurons (C-terminals) and in SVs in nerve terminals in NMJs. The similarity in the distribution of OCT2 in cholinergic neurons and that of vesicular acetyl choline transporter (VAchT), and the fact that OCT2 can transport choline suggest that OCT2 could work as a low-affinity and high-capacity choline transporter at presynaptic terminals in cholinergic neurons in a firing-dependent manner.

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest-derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

1. The effects of essential oils extracted from the wood and leaves of Chamaecyparis obtusa, Cryptomeria japonica and Thujopsis dolabrata on P450s in the hepatic microsomes of the male rat have been investigated. 2. Essential oils did not affect the content of total P450s measured photometrically. However, some metabolic activities and the levels of some forms of P450, including 2B1, were significantly increased, indicating their induction by essential oils. 3. The effects of components derived from essential oils (alpha-pinene, 1,8-cineole, cadinene and borneol) on P450s in rat hepatic microsomes were investigated. The activities of testosterone 2 beta-, 6 beta-, 16 alpha- and 16 beta-hydroxylation and the levels of P4502B1 and 3A2 were increased by 1,8-cineole and cadinene. The induction of P450s by essential oils is thought to be caused by cadinene, a major component. The P450 isoform induced by cadinene is similar to that induced by phenobarbital. However, the magnitude of induction by cadinene was less than that by phenobarbital at the dose levels studied. 4. P4502B1 and 3A2 were induced by essential oils in the perivenous area in the hepatic lobe.

To study the effect of localised secretion of chemokines on tumour growth, the genes for human (hu) interleukin 8 (IL-8), hu-MCP-1 (MCAF), hu-MIP-1 alpha (LD78), murine (mu)-MCP-1 (JE), mu-MIP-1 alpha or mu-MIP-2 were introduced, via mammalian expression vectors, into Chinese hamster ovary (CHO) cells, and the ability of transfected cells to form tumours in vivo was evaluated. The production of hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha by transfected clones did not influence the growth rate in vitro, but drastically suppressed tumour growth when injected subcutaneously (s.c.) into nude mice. However, clones transfected with hu-MCP-1, mu-MCP-1 or mu-MIP-2 did not show any significant difference in growth rate in vivo compared with clones transfected with vector alone. Histological examination of the site of injection of CHO clones transfected with hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha showed predominantly neutrophilic infiltration. These results indicate that chemokines have potent anti-tumour activity when released, even at low doses, at the tumour site, which may be mediated by recruitment and targeting of neutrophilic granulocytes to chemokine-releasing cells. Our studies highlight the potential usefulness of localised chemokine secretion in inducing potent host anti-tumour defensive responses. Images Figure 1 Figure 3

1. The effects of 4-phenylalkylpyridines (chain length of 0-5, 7, 9 and 11 carbon atoms) on the induction of hepatic microsomal P450 and its multiple forms (1A1/2 (1A1, 1A2), 3A2, 2B1/2, 2C6 and 2E1) in the male and female rat have been investigated.2. P450 induction gradually declined with increasing chain length of the 4-phenylalkylpyridines.3. Immunoblot analysis revealed that three pyridine compounds having methylene units of 0, 1 and 2 only induced P4501A2, whereas those having 4, 5 or 7 methylene units only induced 1A1 in the male rat.4. In the female rat, however, we could not observe such a chain length-dependent differential induction of the P4501A subfamily.5. Induced levels of P4503A2 and 2E1 were dependent on total P450 specific content, but 2C6 was increased in a chain length-dependent manner in both sexes.6. These results provide new information on the differential effects of pyridine-containing compounds on P450 induction in the rat.

High-carbohydrate or high-fat diets have been demonstrated to change ghrelin concentrations in plasma; however, there remains a need to clarify the effects of dietary protein on the interaction between circulating GH and ghrelin concentrations in the ruminant. In this study, we investigated the postprandial changes in plasma concentrations of GH and ghrelin and their interactions when wethers were fed either a high-protein (HP; 40% CP) or a low-protein (LP; 10% CP) diet for 2 wk. The wethers were divided into 2 groups and fed once a day for 2 wk in a randomized crossover design. Each diet contained the same level of ME. Blood was collected from the animals at specific times over 24 h to measure hormones and metabolites. Feeding once a day caused a prompt reduction in the GH and ghrelin concentrations regardless of the type of diet that the wethers consumed. The preprandial concentrations (P = 0.04), area under the curve (AUC; P = 0.04), and incremental AUC (iAUC; P = 0.06) for ghrelin in HP-fed wethers were or tended to be greater than those in LP-fed wethers although concentrations for GH were the same for both diets (P = 0.23). In addition, the time it took for the postprandial ghrelin concentrations to recover to the preprandial concentrations was greater in HP-fed wethers than in LP-fed wethers although this was not true for GH concentrations. Similarly, as for ghrelin, postprandial increase (P0.001) and AUC (P = 0.03) for insulin concentration was greater in the HP-fed wethers than in the LP-fed wethers. From these findings, we concluded that dietary proteins (or some other derived metabolites) may dissociate the interaction between plasma concentrations of GH and ghrelin in wethers.

Neuropathic pain is caused by neural damage or dysfunction and neuropathic pain-related symptoms are resistant to conventional analgesics. Neuroinflammation due to the cytokine-chemokine network may play a pivotal role in neuropathic pain. We demonstrate that macrophage inflammatory protein-1β (MIP-1β) participates in neuropathic pain.Mice received partial sciatic nerve ligation (PSL), and tactile allodynia and thermal hyperalgesia were assessed by von Frey test and Hargreaves test, respectively. Agents were administered into the region surrounding the sciatic nerve (SCN).Using reverse transcription polymerase chain reaction, the mRNA expressions of MIP-1β and its receptor (CC-chemokine receptor 5; CCR5) in the injured SCN were up-regulated after PSL. MIP-1β immunoreactivity was localized in macrophages and Schwann cells and increased in the injured SCN on day 1. PSL-induced tactile allodynia on days 4 to 7 was prevented by the administration of MIP-1β neutralizing antibody (anti-MIP-1β; on days 0, 3 and 6). PSL-induced up-regulations of inflammatory cytokine-chemokine mRNAs in the injured SCN were suppressed with anti-MIP-1β treatment on day 7. Administration of CCR5 antagonist, D-ala-peptide T-amide (on days 0, 3 and 6) prevented tactile allodynia and thermal hyperalgesia on days 4 to 14. Single administration of recombinant mouse MIP-1β (rmMIP-1β) elicited tactile allodynia. Moreover, rmMIP-1β increased the mRNA expression of inflammatory mediators in the SCN on day 1 after administration.These results suggest that MIP-1β is a novel key mediator, and the peripheral MIP-1β-CCR5 axis contributes to neuropathic pain. Therefore, investigation of this cascade might be a validated approach for the elucidation of neuropathic pain mechanisms.