Active transport of glucose across the jejunal epithelium decreases with age in broiler chickens.

Intestinal glucose absorption varies with growth; however, the dynamics of these variations has not been yet fully elucidated in broiler chickens. The present study aimed to compare jejunal glucose uptake and maltose digestion in broilers of 2 different ages, i.e., 1- vs. 5 wk old. Oral D-maltose gavage, everted sac, and Ussing chamber experiments were carried out to investigate intestinal glucose absorption and mRNA expression of glucose-transport-related genes as well as jejunal maltase activity. Upon gavage, glucose concentrations peaked at 10 min post-administration in 1-wk-old chicks, while they peaked at 40 min in 5-wk-old chickens. Glucose concentrations at 10 min were significantly higher in the 1-wk-old chicks (P = 0.010). Using the everted sacs experimental setup, 5 intestinal regions i.e., duodenum, proximal jejunum, distal jejunum, proximal ileum, and distal ileum, were targeted to examine D-maltose digestion and glucose transport across the intestinal mucosa. In the distal and proximal ileum, glucose concentrations were found to be significantly higher in the serosal compartment of the 1-wk-old chicks upon incubation with D-maltose (25 mM) (P < 0.05), while in the mucosal compartment the levels were significantly higher in the 5-wk-old chickens (P < 0.05). An Ussing chamber setup was employed to measure glucose-induced short-circuit current (ΔIsc) in the mucosal epithelium of the jejunum. In response to the addition of D-maltose (10 mM) into the mucosal compartment, ΔIsc was significantly higher in the 1-wk-old chicks (P = 0.018). Furthermore, no variations in jejunal maltase activity were observed between the 2 age groups. While jejunal glucose absorption was lower in the 5-wk-old chickens, the mRNA expression levels of jejunal SGLT1, GLUT2, and Na+/K+-ATPase did not show any significant differences between the 2 age groups. Our results suggest that the active transport of glucose across the jejunal epithelium decreases upon growth in broiler chickens but is not accompanied by any variations in maltase activity or in the expression of glucose-absorption-related genes.

Short communication: opposing effects of lactoferrin on the proliferation of fibroblasts and epithelial cells from bovine mammary gland.

Lactoferrin is present in several physiologic fluids, including milk and colostrum. Recently, evidence has accumulated that lactoferrin acts as a regulator of cell proliferation. Lactoferrin mRNA and protein levels in bovine mammary glands are known to markedly increase after cessation of milking. To clarify the role of bovine lactoferrin (bLF) in mammary involution and remodeling during dry periods, we investigated whether bLF affects the proliferation of cultured cells derived from bovine mammary gland and examined the mechanism underlying the proliferative response to bLF. Addition of bLF to the culture medium increased the proliferation of bovine mammary stromal fibroblasts (bMSF), but decreased that of bovine mammary epithelial cells (bMEC). Proliferation was significantly increased in the bMSF treated with bLF (100µg/mL or greater) as compared with unstimulated cells. The maximal proliferative effect of bLF on bMSF occurred at 1,000µg/mL, such that the proliferation of the bLF-stimulated bMSF was approximately 2.5 times that of unstimulated cells. The bLF increased the production of proliferating cell nuclear antigen and rapid phosphorylation of the p44/p42 mitogen-activated protein kinase in bMSF, but not in bMEC. The bLF-induced proliferation and production of proliferating cell nuclear antigen in bMSF was suppressed by U0126, a specific inhibitor of mitogen-activated protein kinase. Furthermore, treatment with bLF for 24h decreased the mRNA levels of the 3 isoforms of transforming growth factor ß in bMSF (16-66%) but upregulated those in bMEC (122-157%). These opposite effects of bLF on the proliferation of epithelial and fibroblast cells and their expression of transforming growth factor ß may play a crucial role in bovine mammary involution and remodeling.

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.

Proposal of a simplified technique for staining bacterial spores without applying heat--successful modification of Moeller's method.

BACKGROUND AND AIMS: As the bacterial spores are difficult to stain, a number of staining techniques including their modifications have been proposed to date. Most of the conventional staining procedures unexceptionally contain the step of staining with steamed dye reagent in order to increase the stainability of the spores. We made an attempt to improve the conventional Moeller's methods for staining bacterial spores. METHODS: Spores of Bacillus species were stained with our modified Moeller's spore stain and evaluated for its staining properties. We investigated the stainability of both of the conventional and the modified Moeller's methods and the evaluation was made whether or not the step of steaming of Kinyoun's carbol-fuchsine dye reagent could be omitted by adding to aliquots of Tergitol 7, in place of the conventional dye solution steamed for some interval over hot blue flame of a Bunsen burner. RESULTS: We successfully omitted the heating step of steaming the Kinyoun's carbol fuchsine dye solution in the Moeller's method of bacterial spore stain, by the replacement of Kinyoun's carbol-fuchsine dye solution involving 2 drops of Tergitol 7, nonionic polyglycol ether surfactants type NP-7 (Sigma-Aldrich Japan, Tokyo, Japan) per 10 ml of Kinyoun's carbol-fuchsine dye solution. Bacillus spores stained pink to red and vegetative bacterial cells stained blue, although without applying any heating step during the whole course of staining processes including the fixation process. The novel staining method of our proposal resulted in far better satisfactory stainability in comparison with the conventional Moeller's method with the steaming dye solution. CONCLUSIONS: The modified spore stain without applying any heating step using the Kinyoun's carbol-fuchsine dye solution with an addition of Tergitol 7 aliquots was demonstrated to be reproducible and yielded consistent and satisfactory stainability. This simplified staining procedure is rapid to perform and found to be applicable to detect the bacterial spores in routine clinical microbiology laboratories.