Deciphering the origin of Aspergillus flavus NRRL21882, the active biocontrol agent of Afla-Guard®.

Single nucleotide polymorphisms (SNPs) of genome sequences of eight Aspergillus flavus and seven Aspergillus oryzae strains were extracted with Mauve, a multiple-genome alignment programme. A phylogenetic analysis with sequences comprised of concatenated total SNPs by the unweighted pair group method with arithmetic mean (UPGMA) of MAFFT adequately separated them into three groups, A. flavus S-morphotype, A. flavus L-morphotype and A. oryzae. Divergence time inferred for A. flavus NRRL21882, the active agent of the biocontrol product Afla-Guard® , and S-morphotype was about 5·1 mya. Another biocontrol strain, A. flavus AF36, diverged from aflatoxigenic L-morphotype about 2·6-3·0 mya. Despite the close relatedness of A. oryzae to A. flavus, A. oryzae strains likely evolved from aflatoxigenic Aspergillus aflatoxiformans (=A. parvisclerotigenus). A survey of A. flavus populations implies that prior Afla-Guard® applications are associated with prevalence of NRRL21882-type isolates in Mississippi fields. In addition, a few NRRL21882 relatives were identified. A. flavus Og0222, a biocontrol ingredient of Aflasafe™, was verified as a NRRL21882-type strain, having identical sequence breakpoints that led to deletion of aflatoxin and cyclopiazonic acid gene clusters. A similar UPGMA analysis suggests that the occurrence of NRRL21882-type strains is a more recent event.

Effects of replacement of para-grass with oil palm compounds on body weight, food intake, nutrient digestibility, rumen functions and blood parameters in goats.

OBJECTIVE: The aim of the present study was to investigate the beneficial effects of dietary supplementation with oil palm frond (leaf) (OPF) with and without oil palm meal (OPM) on nutrient intake and digestibility, ruminal fermentation and growth performance in goats. METHODS: Six female crossbred goats were fed for 28 days of 3 diet treatments; 100% paragrass (T1); 50% para-grass + 50% OPF (T2), and 30% para-grass + 50% OPF + 20% OPM (T3). Body weight, rectal temperature, respiratory rate, and urine volume, food intake, dry matter intake and water intake were measured daily. Nutrient digestibility was determined from five consecutive days of last week in each diet. Ruminal fluid, urine and blood were collected at the end for determination of rumen protozoa and volatile fatty acid contents, urinary allantoin excretion, blood cell count and chemistry profiles. RESULTS: Goats fed T2 and T3 showed higher dry matter and nutrients intakes while protein digestibility was suppressed compared with those for T1. Crude fat digestibility declined in T2 but maintained after adding the OPM (T3). High fat intake by giving OPF and OPM corresponded to a higher ruminal acetate/propionate ratio (C2/C3) and serum cholesterol level. An increased urinary allantoin/creatinine ratio was found in T2 and T3 compared with T1, implying an increased number of ruminal microbes. CONCLUSION: Increased dry matter intake in T2 and T3 suggested that oil palm by-products are partly useful as a replacement for para-grass in goats. Replacement with the by-products increased plasma cholesterol level, which suggested that these products are a useful energy source. Changes in rumen parameters suggested an increased microbial number and activity suitable for acetate production. However, the limited digestibility of protein implies that addition of high protein feeds may be recommended to increase body weight gain of goats.

Changes in the expression of α-tocopherol-related genes in liver and mammary gland biopsy specimens of peripartum dairy cows.

Blood α-tocopherol (α-Toc) concentrations decline gradually throughout the prepartum period, reaching the nadir after calving in dairy cows. The 6 α-Toc-related molecules [α-Toc transfer protein (TTPA); afamin; scavenger receptor class B, Type I; ATP-binding cassette transporter A1; tocopherol-associated protein (SEC14L2); and cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2)] are expressed in liver and other peripheral tissues. These molecules could regulate α-Toc transport, blood concentrations, and metabolism of α-Toc. Therefore, the aim of this study was to evaluate the changes in the expression of α-Toc-related genes in liver and mammary gland tissues of dairy cows around calving, which have remained elusive until now. In experiment (Exp.) 1, 28 multiparous Holstein cows were used (from -5 to 6 wk relative to parturition) to monitor the changes in dietary α-Toc intake, blood concentrations of α-Toc, and lipoproteins; in Exp. 2, 7 peripartum Holstein cows were used (from -4 to 4 wk relative to parturition) for liver tissue biopsy; and in Exp. 3, 10 peripartum Holstein cows were used (from -8 to 6 wk relative to parturition) to carry out the mammary gland tissue biopsy and milk sampling. In Exp. 1, the serum α-Toc concentrations declined gradually with decreasing amount of α-Toc intake and plasma high-density lipoprotein concentrations toward calving time. However, in the early lactation period after calving, serum α-Toc concentrations remained at a lower concentration despite the recovery of α-Toc intake and plasma high-density lipoprotein concentrations. In Exp. 2, just after calving, the TTPA, SEC14L2, afamin, and albumin mRNA expression levels in the liver were temporarily downregulated, and the hepatic mRNA levels of endoplasmic reticulum stress-induced unfolded protein response markers and acute-phase response marker increased at calving. In Exp. 3, the concentrations of α-Toc in colostrum were greater than those in precolostrum (samples were collected at wk -1 relative to parturition) and mature milk. The expression of TTPA, SEC14L2, and CYP4F2 mRNA in bovine mammary gland tissue was detected. However, TTPA and SEC14L2 mRNA expressions showed the opposite trends: the expression levels of TTPA mRNA peaked whereas SEC14L2 mRNA reached a nadir at calving. These results indicate that the expression of α-Toc-related genes involved in specific α-Toc transfer and metabolism in the liver and mammary gland are altered during calving. Moreover, these changes might be associated with the maintenance of lower serum α-Toc concentrations after calving.

Postweaning changes in the expression of chemerin and its receptors in calves are associated with the modification of glucose metabolism.

Chemerin, originally known as a chemoattractant derived from adipose tissue and the liver, has been reported to have regulatory functions in gluconeogenesis, peripheral insulin sensitivity, and insulin secretion. This study was conducted to assess the postweaning changes in expression of this cytokine and its physiological role in the modification of glucose metabolism associated with weaning. Eighteen tissue samples were collected from Holstein calves (90 d of age; n = 4) to investigate the tissue distributions of chemerin and its receptors genes. was highly expressed in the liver, and secreted chemerin protein was found in the plasma. Among the receptors of chemerin, and were ubiquitously expressed whereas was predominantly expressed in the liver. The changes in glucose metabolism and expression of these genes after weaning were assessed by comparing suckling calves (n = 6) and weaned calves (n = 8) of Japanese Black cattle. No significant difference was observed in plasma glucose levels between suckling and weaned calves (P = 0.22), whereas the plasma level of total ketone bodies was significantly higher in weaned calves (P < 0.01). Plasma levels of insulin and cortisol did not differ between suckling and weaned calves. The mRNA levels of certain key enzymes involved in hepatic gluconeogenesis were also altered; for instance, level was lower in postweaning calves (P < 0.05) and () level tended to be higher after weaning (P = 0.08). However, was not altered after weaning. The plasma levels of hepatic stress indicators were also changed, with aspartate transaminase, alanine transaminase, and lactate dehydrogenase being significantly elevated in postweaning calves (P < 0.05). Chemerin protein in liver tissue was less abundant in weaned calves (P < 0.05), although there were no changes in its transcript levels. The abundance of plasma chemerin protein did not change after weaning (P = 0.95). In summary, these data indicate that as a consequence of weaning, which causes physiological stress and alters hepatic metabolism, chemerin protein expression within the liver is downregulated, indicating that chemerin plays a role in the upregulation of hepatic expression via its inhibitory effect on hepatic gluconeogenesis.

Expression of α-tocopherol-associated genes and α-tocopherol accumulation in Japanese Black (Wagyu) calves with and without α-tocopherol supplementation.

The aim of the study was to clarify 1) the distribution of 6 α-tocopherol (α-Toc)-associated gene expressions in 20 major tissues, including metabolic, reproductive, endocrine, immune, and digestive and absorptive tissues, in relation to α-Toc status and 2) the change in expression patterns of the genes induced when α-Toc was orally administered to Japanese Black (JB) calves. This study examined weaned male JB calves ( = 10), of which 5 calves were orally administered α-Toc for 2 wk (30 IU·kg BW·d; TOC group). The others did not receive the α-Toc supplement and were the control (CONT) group. The 20 tissues and venous blood (serum) were sampled on the final day. In both groups, the mean mRNA expression levels for α-Toc transfer protein, afamin (AFM), ATP-binding cassette transporter A1, and tocopherol-associated protein were greatest in the liver ( < 0.05), whereas scavenger receptor class B, Type I (SR-BI) mRNA was greatest in the adrenal gland ( < 0.05). The gene for cytochrome P450 family 4, subfamily F, polypeptide 2 was most highly expressed in the liver, testes, and adrenal gland. The α-Toc content was greatest ( < 0.05) in the testes of the 20 sampled tissues in the CONT group. However, the levels in the testes and jejunum were similar and greater ( < 0.05) than the levels in the other 18 tissues in the TOC group. The mean increase in α-Toc levels after oral α-Toc administration (mean α-Toc content for the TOC group divided by the CONT group content) were greater ( < 0.05) in the jejunum (40.7-fold) and duodenum and liver (26.3- and 23.1-fold) than in the serum (7.8-fold). In the liver, α-Toc administration significantly increased ( < 0.05) the AFM and SR-BI mRNA expression levels. The results show that the liver may play an important role in the regulation of α-Toc disposition, but other peripheral tissues that accumulate large amounts of α-Toc could moderate the local α-Toc status and functions, as inferred from the high expressions of the α-Toc-associated genes in JB calves.

The regulation of oxytocin receptor gene expression during adipogenesis.

Although it has been reported that oxytocin stimulates lipolysis in adipocytes, changes in the expression of oxytocin receptor (OTR) mRNA in adipogenesis are still unknown. The present study aimed to investigate the expression of OTR mRNA during adipocyte differentiation and fat accumulation in adipocytes. OTR mRNA was highly expressed in adipocytes prepared from mouse adipose tissues compared to stromal-vascular cells. OTR mRNA expression was increased during the adipocyte differentiation of 3T3-L1 cells. OTR expression levels were higher in subcutaneous and epididymal adipose tissues of 14-week-old male mice compared to 7-week-old male mice. Levels of OTR mRNA expression were higher in adipose tissues at four different sites of mice fed a high-fat diet than in those of mice fed a normal diet. The OTR expression level was also increased by refeeding for 4 h after fasting for 16 h. Oxytocin significantly induced lipolysis in 3T3-L1 adipocytes. In conclusion, a new regulatory mechanism is demonstrated for oxytocin to control the differentiation and fat accumulation in adipocytes via activation of OTR as a part of the hypothalamic-pituitary-adipose axis.

Increased plasma ghrelin suppresses insulin release in wethers fed with a high-protein diet.

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 ghrelin O-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.

Genetic polymorphisms and protein structures in growth hormone, growth hormone receptor, ghrelin, insulin-like growth factor 1 and leptin in Mehraban sheep.

The somatotropic axis, the control system for growth hormone (GH) secretion and its endogenous factors involved in the regulation of metabolism and energy partitioning, has promising potentials for producing economically valuable traits in farm animals. Here we investigated single nucleotide polymorphisms (SNPs) of the genes of factors involved in the somatotropic axis for growth hormone (GH1), growth hormone receptor (GHR), ghrelin (GHRL), insulin-like growth factor 1 (IGF-I) and leptin (LEP), using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods in 452 individual Mehraban sheep. A nonradioactive method to allow SSCP detection was used for genomic DNA and PCR amplification of six fragments: exons 4 and 5 of GH1; exon 10 of GH receptor (GHR); exon 1 of ghrelin (GHRL); exon 1 of insulin-like growth factor-I (IGF-I), and exon 3 of leptin (LEP). Polymorphisms were detected in five of the six PCR products. Two electrophoretic patterns were detected for GH1 exon 4. Five conformational patterns were detected for GH1 exon 5 and LEP exon 3, and three for IGF-I exon 1. Only GHR and GHRL were monomorphic. Changes in protein structures due to variable SNPs were also analyzed. The results suggest that Mehraban sheep, a major breed that is important for the animal industry in Middle East countries, has high genetic variability, opening interesting prospects for future selection programs and preservation strategies.

Proliferating bovine intramuscular preadipocyte cells synthesize leptin.

Leptin is thought to be not only a satiety factor but also a stimulator of angiogenesis. We examined leptin, PPARγ2, and vascular endothelial growth factor (VEGF) expression in bovine intramuscular preadipocyte (BIP) cells during proliferation. The cells were seeded at 0.85 × 10(4) cells/cm(2) and collected every day until the fifth day after passage. Leptin mRNA was present in the cells between days 2 and 4, as indicated by RT-PCR analysis. Western blot analysis showed a band for leptin at approximately 16 kDa on all of the days during growth, and the cytoplasmic concentration of leptin was highest on day 2 and decreased gradually thereafter. A PPARγ2 band at approximately 54 kDa was also observed on all days. The concentration was highest on day 2 and decreased thereafter, which is similar to the expression pattern of leptin. In constant, the expression level of VEGF protein did not change while in culture. We have demonstrated that BIP cells can synthesize both leptin and PPARγ2, with maximal synthesis occurring during maximal proliferation. Given the role of leptin in angiogenesis, we speculate that leptin is involved in the neovascularization of adipose tissue, because new organization of adipose tissue requires the growth of new blood vessels.

Morphological assessment of the luminal surface of olfactory epithelium in mice deficient in tissue plasminogen activator following bulbectomy.

OBJECTIVE: This study aimed to investigate the function of tissue plasminogen activator in the olfactory epithelium of mice following neural injury. METHOD: Transmission electron microscopy was used to study the changes in the morphology of the olfactory epithelium 1-7 days after surgical ablation of the olfactory bulb (bulbectomy). RESULTS: Prior to bulbectomy, a uniformly fine material was observed within some regions of the olfactory epithelium of mice deficient in tissue plasminogen activator. At 2-3 days after bulbectomy, there were degenerative changes in the olfactory epithelium. At 5-7 days after bulbectomy, we noted drastic differences in olfactory epithelium morphology between mice deficient in tissue plasminogen activator and wild-type mice (comparisons were made using findings from a previous study). The microvilli seemed to be normal and olfactory vesicles and receptor neuron dendrites were largely intact in the olfactory epithelium of mice deficient in tissue plasminogen activator. CONCLUSION: The tissue plasminogen activator plasmin system may inhibit the regeneration of the olfactory epithelium in the early stages following neural injury.

A high-protein diet induces dissociation between plasma concentrations of growth hormone and ghrelin in wethers.

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 (P < 0.001) 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.

Contributions of FASN and SCD gene polymorphisms on fatty acid composition in muscle from Japanese Black cattle.

The fatty acid synthase (FASN) and stearoyl-CoA desaturase (delta-9-desaturase) (SCD) genes affect fatty acid composition. This study evaluated the contributions of polymorphisms of these genes on fatty acid composition in muscle in two different populations: 1189 and 1058 Japanese Black cattle from the Miyagi and the Yamagata populations respectively. We sampled intramuscular fat from the longissimus thoracis muscle in the Miyagi population and from the trapezius muscle in the Yamagata population. The collective contributions of FASN and SCD polymorphisms to total additive genetic variance for oleic acid were 13.46% in the Miyagi population and 16.29% in the Yamagata population and to phenotypic variance were 5.45% and 6.54% respectively. Although the individual effects of FASN and SCD polymorphisms on fatty acid composition were small, overall gene substitution may effectively improve fatty acid composition. In addition, we found that gene polymorphism contributions of fatty acids varied by population even in the same breed.

Apelin is involved in postprandial responses and stimulates secretion of arginine-vasopressin, adrenocorticotropic hormone, and growth hormone in the ruminant.

Apelin and its mRNA are expressed in several tissues, including the supraoptic and paraventricular nuclei in the hypothalamus. Although apelin is reported to be involved in the regulation of fluid homeostasis, little is known about the postprandial dynamics of apelin in plasma and its regulatory effects on the anterior pituitary hormones of ruminants. Therefore, the aims of this study were to investigate the following: (1) changes in plasma apelin concentrations in response to food intake under conditions of hydration (free access to water) or dehydration (water restriction), and (2) the effects of intravenous administration of apelin on plasma concentrations of arginine-vasopressin (AVP), ACTH, GH, and insulin. In Experiment 1 with the use of goats, the postprandial plasma apelin concentration was significantly increased under the dehydration condition compared with the hydration condition, and this increase was accompanied by increased plasma concentrations of AVP and ACTH after 24 h of dehydration. In Experiment 2 with the use of sheep and hydration conditions, the intravenous administration of apelin ([Pyr(1)]-apelin-13; 0.5 mg/head) caused a tendency to increase or caused a significant increase in plasma concentrations of AVP, ACTH, GH, insulin, and glucose. On the basis of these findings, we concluded that apelin is involved in the feeding process, and it regulates endocrine functions in the anterior pituitary gland via AVP in ruminant animals.

The Regulation of Chemerin and CMKLR1 Genes Expression by TNF-α, Adiponectin, and Chemerin Analog in Bovine Differentiated Adipocytes.

Adipokines, adipocyte-derived protein, have important roles in various kinds of physiology including energy homeostasis. Chemerin, one of adipocyte-derived adipokines, is highly expressed in differentiated adipocytes and is known to induce macrophage chemotaxis and glucose intolerance. The objective of the present study was to investigate the changes of chemerin and the chemokine-like-receptor 1 (CMKLR1) gene expression levels during differentiation of the bovine adipocyte and in differentiated adipocytes treated with tumor necrosis factor-α (TNF-α), adiponectin, leptin, and chemerin (peptide analog). The expression levels of the chemerin gene increased at d 6 and 12 of the differentiation period accompanied by increased cytoplasm lipid droplets. From d 6 onward, peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene expression levels were significantly higher than that of d 0 and 3. In contrast, CMKLR1 expression levels decreased at the end of the differentiation period. In fully differentiated adipocytes (i.e. at d 12), the treatment of TNF-α and adiponectin upregulated both chemerin and CMKLR1 gene expression levels, although leptin did not show such effects. Moreover, chemerin analog treatment was shown to upregulate chemerin gene expression levels regardless of doses. These results suggest that the expression of chemerin in bovine adipocyte might be regulated by chemerin itself and other adipokines, which indicates its possible role in modulating the adipokine secretions in adipose tissues.

Cloning, expression analysis, and regulatory mechanisms of bovine chemerin and chemerin receptor.

Recently, we reported that chemerin, a new adipokine, is highly expressed in the adipose tissue, up-regulated during adipocyte differentiation, and regulates adipogenesis via its own receptor in mice. The objectives of this study were to clone chemerin and its receptor from the adipose tissues of Japanese Black cattle and to investigate the expression of these genes in 16 different tissues. We compared the gene expression of chemerin and its receptor between adipocytes and stromal-vascular (S-V) cells (non-adipocytes) prepared from subcutaneous adipose tissue. In addition, we investigated the mRNA expression levels of chemerin and its receptor in bovine differentiated adipocytes. The DNA sequences of bovine chemerin and its receptor were determined, and they were found to be highly homologous to those of humans, mice, and pigs. The amino acid sequences predicted for the full-length cDNA of bovine chemerin and its receptor were also similar to those of humans, mice, and pigs, suggesting that these genes have similar functions. Bovine chemerin mRNA was highly expressed in the adipose and liver tissues, and the transcripts of chemerin receptor were widely expressed in several tissues including adipose, muscle, liver, and brain tissues. The expression of bovine chemerin mRNA was higher in adipocytes than in S-V cells prepared from adipose tissue. The transcripts of chemerin and its receptor were up-regulated during adipocyte differentiation. Treatment with tumor necrosis factor (TNF)-alpha (10 ng/mL) in bovine differentiated adipocytes increased the mRNA expression of chemerin and its receptor. These results indicate that chemerin, a new adipokine highly expressed in the adipocytes of bovine adipose tissue, is the TNF-alpha-up-regulated gene with a role in adipogenesis.

Plasma hormone and metabolite concentrations involved in the somatotropic axis of Japanese Black heifers in association with growth hormone gene polymorphism.

Bovine growth hormone (bGH) gene polymorphism of leucine (Leu)-threonine (Thr) (allele A), valine (Val)-Thr (allele B), and Val-methionine (Met) (allele C) at codons 127 and 172 was shown to relate with carcass trait variations in Japanese Black cattle. In this study, 10-mo-old Japanese Black heifers with growth hormone (GH) genotypes AA, AB, BB, AC, BC, and CC (N=141) were compared for basal GH, insulin-like growth factor-1 (IGF-1), insulin, ghrelin, glucose, and nonesterified fatty acid (NEFA) concentrations. Growth hormone release was also measured as response to growth hormone-releasing hormone (GHRH) (0.4 microg/kg body weight [BW]) using 18 heifers with GH genotypes AA, BB, and CC (n=6 for each group). The genotype AA heifers showed the greatest BW among genotypes (P<0.05). Genotype AC, BC, and CC heifers showed greater GH concentrations than genotype AA, AB, or BB heifers, in which genotype CC heifers had the highest concentrations (P<0.05). However, IGF-1 concentrations did not significantly differ. The genotype AA and BB heifers had a greater GH release at 60 min following GHRH injection than did the genotype CC heifers. The area under the curve (AUC; P<0.07) and incremental area (IA; P<0.08) of GH responses to the GHRH challenge tended to be the highest in the genotype AA heifers and the lowest in the genotype CC heifers. In conclusion, GH gene polymorphism altered GH, which may have contributed to differences in BW and carcass traits among genotypes.

The effect of Bacillus subtilis mouth rinsing in patients with periodontitis.

Bacillus subtilis is an effective probiotic product for prevention of enteric infections both in humans and animals. We hypothesized that a mouth rinse containing Bacillus subtilis should adhere to and colonize part of the oral bacteria on periodontal tissue. The rinsing ability of Extraction 300E (containing Bacillus subtilis: E-300) was compared with that of a mouth wash liquid , Neosteline Green (benzethonium chloride; NG) that is commonly used in Japan. Compared with NG rinsing, E-300 rinsing resulted in a marked change in the BANA-score. The mean BANA values (score +/- SD) over the course of the study from 0 to 30 days were 1.52 +/- 0.51 (p < or = 0.1) and 0.30 +/- 0.47 (p < or = 0.01) for E-300, and 1.56 +/- 0.51 and 0.93 +/- 0.68 for NG, respectively. Gingival Index also had improvement, while probing pocket depth and bleeding on probing showed small improvements. Mouth rinsing with E-300 significantly reduced periodontal pathogens compared with NG. These results suggest that Bacillus subtilis is an appropriate mouth rinse for patients with periodontitis.

Genetic associations of residual feed intake with serum insulin-like growth factor-I and leptin concentrations, meat quality, and carcass cross sectional fat area ratios in Duroc pigs.

Genetic relationships of measures of residual feed intake and daily feed intake with serum IGF-I concentrations at 8 wk of age and at 105 kg of BW, serum leptin concentration at 105 kg of BW, meat quality, and different fat accumulation traits on 834 Duroc pigs in 7 generations were estimated. Two measures of residual feed intake were estimated from the differences between actual and predicted feed intake: phenotypic residual feed intake (RFI(phe)) and nutritional residual feed intake (RFI(nut)). Meat quality traits included drip loss, cooking loss, pork color score, pork lightness (L*), and pH, whereas fat accumulation traits were subcutaneous fat, intermuscular fat, and total fat percent at 5-6th thoracic vertebra; subcutaneous fat, intermuscular fat, abdominal fat, and total fat percent at one-half body length and at last thoracic vertebra, and seam fat score. The IGF-I concentrations at 8 wk of age and 105 kg of BW had weak genetic correlations with measures of residual feed intake and daily feed intake (absolute values ranging from 0.14 to 0.24). The genetic correlations between measures of residual feed intake and serum leptin concentration were strong and positive (r(g) with RFI(phe) and RFI(nut) were 0.74 and 0.80, respectively). Residual feed intake was moderately but negatively correlated with cooking loss (r(g) with RFI(phe) and RFI(nut) were -0.42 and -0.49, respectively), whereas daily feed intake was moderately and positively correlated with drip loss and pH (0.33 and 0.36, respectively). Daily feed intake was also moderately correlated with subcutaneous fat accumulations at the 5-6th thoracic vertebra (0.31) and one-half body length (0.31) regions and was strongly correlated with accumulations at the last thoracic vertebra region (0.57). The genetic correlations between daily feed intake and intermuscular fat accumulations at all of the carcass sites were strong (0.60, 0.76, and 0.56 for intermuscular fat at 5-6th thoracic vertebra, one-half body length, and last thoracic vertebra, respectively). Residual feed intake was strongly and positively correlated with all of the fat accumulation traits (ranging from 0.53 to 0.88). The results indicate that reducing residual feed intake (increased efficiency) would lead to increased cooking loss and darkness, and decreased serum leptin concentration, fat accumulations at the different sites, and seam fat at the 6th rib interface of pork carcasses.

[Thymic carcinoma involving aortic arch; report of a case].

Adenocarcinoma of the thymus is a very rare malignant tumor. The standard treatment for advanced thymic carcinoma has not yet been established, and the prognosis is poor. We report a case of thymic carcinoma that involving the aortic arch and the innominate vein. A 78-year-old woman was admitted to our hospital complaining of hoarseness in April 2007. The computed tomography (CT) scan showed an anterior mediastinal tumor contiguous to the aortic arch and the innominate vein with swelling lymphnodes. Microspcopic examinations of specimens obtained by CT-guided needle biopsy revealed poorly differenciated adenocarcinoma. The carcinoembryonic antigen (CEA) level of serum elevated at 54.9 ng/ml. Thymic carcinoma was diagnosed. The chemoradiotherapy [concurrent, carboplatin (CBDCA) + paclitaxel(TXL)-->vinorelbine (NVB), 60 Gy] was performed, but the effect of the therapy was limited. The resection of the tumor with a part of aortic arch and other peripheral tissues was performed in Augast 2007. The postoperative course was uneventful and the CEA level of serum lowered to the normal. She was discharged 30 days after surgery.

Microwave irradiation for fixation and immunostaining of endothelial cells in situ.

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.