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.

Relationships between conception rate in Holstein heifers and cows and milk yield at various stages of lactation.

We investigated the relationships between conception rates (CRs) at first service in Japanese Holstein heifers (i.e. animals that had not yet had their first calf) and cows and their test-day (TD) milk yields. Data included records of artificial insemination (AI) for heifers and cows that had calved for the first time between 2000 and 2008 and their TD milk yields at 6 through 305 days in milk (DIM) from first through third lactations. CR was defined as a binary trait for which first AI was a failure or success. A threshold-linear animal model was applied to estimate genetic correlations between CRs of heifers or cows and TD milk yield at various lactation stages. Two-trait genetic analyses were performed for every combination of CR and TD milk yield by using the Bayesian method with Gibbs sampling. The posterior means of the heritabilities of CR were 0.031 for heifers, 0.034 for first-lactation cows and 0.028 for second-lactation cows. Heritabilities for TD milk yield increased from 0.324 to 0.433 with increasing DIM but decreased slightly after 210 DIM during first lactation. These heritabilities from the second and third lactations were higher during late stages of lactation than during early stages. Posterior means of the genetic correlations between heifer CR and all TD yields were positive (range, 0.082 to 0.287), but those between CR of cows and milk yields during first or second lactation were negative (range, -0.121 to -0.250). Therefore, during every stage of lactation, selection in the direction of increasing milk yield may reduce CR in cows. The genetic relationships between CR and lactation curve shape were quite weak, because the genetic correlations between CR and TD milk yield were constant during the lactation period.

Different responses in postprandial plasma ghrelin and GH levels induced by concentrate or timothy hay feeding in wethers.

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.

Ephrin-A1 promotes the malignant progression of intestinal tumors in Apc(min/+) mice.

The ephrin-A1 and EphA receptors are frequently highly expressed in different human cancers, suggesting that they may promote tumor development and progression. We generated transgenic mice carrying Fabpl(4xat-132) ephrin-A1, which express ephrin-A1 in the intestinal epithelial cells. Those mice were then mated with Apc(min/+) mice to produce the compound mice, which overexpress ephrin-A1 in the intestinal tumors of Apc(min/+) mice. We compared the number, size and histopathological features of the intestinal tumors in the Fabpl(4xat-132) ephrin-A1/Apc(min/+) compound mice with those of the Apc(min/+) mice. The compound mice showed an increased number of intestinal tumors, significantly in the large intestine, and developed more invasive tumors. Among the 20 mice of each type examined, 5 Apc(min/+) mice developed 5 invasive tumors, 1 invasive tumor in each mouse, in the proximal or middle portions of the small intestine. On the other hand, 14 out of 20 compound mice developed 29 invasive tumors and 16 of them were in the distal small intestine and the large intestine, where transgenic ephrin-A1 was highly expressed. These results suggested that the increased expression of ephrin-A1 accelerated the malignant progression of the intestinal adenoma to invasive tumors.

Expression of matrilysin (matrix metalloproteinase-7) in primary cutaneous and metastatic melanoma.

BACKGROUND: Matrilysin (MMP-7), a member of the matrix metalloproteinase (MMP) family of proteins, is expressed in various types of malignant tumours. There have been no previous studies of the correlation between matrilysin expression and melanoma. OBJECTIVES: Protein expression of matrilysin was evaluated in human cutaneous melanomas, metastatic melanomas, acquired common melanocytic naevi and Spitz naevi, and the data were corrected with the clinicopathological factors. METHODS: We retrospectively investigated 18 primary melanomas, 15 metastatic melanomas, 10 common melanocytic naevi and five Spitz naevi samples at our clinic using immunohistochemistry (IHC). Both promatrilysin and active matrilysin were found in the melanoma tissue extracts by Western immunoblotting. In situ hybridization demonstrated that melanoma cells selectively express matrilysin mRNA. RESULTS: Of the melanoma samples, 29 of 33 (87 x 9%) were positive for matrilysin, including 14 of 18 (77 x 8%) primary cutaneous melanomas and 15 of 15 (100%) metastatic melanomas. In contrast, matrilysin was not expressed in common naevi or Spitz naevi. The matrilysin IHC staining score in primary melanomas was associated with the presence of metastases, tumour thickness and TNM staging (P=0 x 001, 0 x 025 and 0 x 021, respectively). The 5-year overall survival was 26.3% for matrilysin-positive cases and 100% for matrilysin-negative cases among melanoma specimen. CONCLUSIONS: We found matrilysin expression in primary melanomas and in metastatic melanomas. We further demonstrated that the matrilysin IHC staining score was associated with invasive depth of primary melanoma lesions and metastases. Our observations indicate that matrilysin may be associated with melanoma progression, and may enhance melanoma tumour cell invasion. Therefore, matrilysin may be potentially valuable as a prognostic indicator to predict the clinical behaviour of melanoma.

Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer.

Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (beta-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear beta-catenin accumulation (13/15; 87%) and detected the active form of beta-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%), and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.

ELAC2, a putative prostate cancer susceptibility gene product, potentiates TGF-beta/Smad-induced growth arrest of prostate cells.

Transforming growth factor-beta (TGF-beta) elicits a potent growth inhibitory effect on many normal cells by binding to specific serine/threonine kinase receptors and activating specific Smad proteins, which regulate the expression of cell cycle genes, including the p21 cyclin-dependent kinase (CDK) inhibitor gene. Interestingly, cancer cells are often insensitive to the anti-mitogenic effects of TGF-beta for which the molecular mechanisms are not well understood. In this study, we found that the candidate prostate cancer susceptibility gene ELAC2 potentiates TGF-beta/Smad-induced transcriptional responses. ELAC2 associates with activated Smad2; the C-terminal MH2 domain of Smad2 interacts with the N-terminal region of ELAC2. Small interfering siRNA-mediated knock-down of ELAC2 in prostate cells suppressed TGF-beta-induced growth arrest. Moreover, ELAC2 was shown to specifically associate with the nuclear Smad2 partner, FAST-1 and to potentiate the interaction of activated Smad2 with transcription factor Sp1. Furthermore, activation of the p21 CDK inhibitor promoter by TGF-beta is potentiated by ELAC2. Taken together our data indicate an important transcriptional scaffold function for ELAC2 in TGF-beta/Smad signaling mediated growth arrest.

Successful treatment of hepatitis B virus-associated membranous nephropathy with lamivudine.

We present a case of chronic hepatitis B with membranous nephropathy, that was improved by lamivudine treatment. A 37-year-old man was admitted to our hospital for the evaluation of proteinuria. He was diagnosed as having chronic glomerulonephritis associated with chronic hepatitis B. Histopathological findings of the renal biopsy specimen indicated membranous nephropathy. He suffered from nephrotic syndrome associated with leg edema, which was parallel to the exacerbation of hepatitis. Lamivudine was started for the treatment of hepatitis, which caused the disappearance of serum hepatitis B virus DNA and the normalization of ALT level in 4 weeks. Additionally, proteinuria disappeared 120 weeks after the treatment was started. Lamivudine treatment may remit HBV-associated nephropathy.

Changes in gene expression of glucose transporters in lactating and nonlactating cows.

Glucose delivery and uptake by the mammary gland are a rate-limiting step in milk synthesis. It is thought that insulin-independent glucose uptake decreases in tissues, except for the mammary gland, and insulin resistance in the whole body increases following the onset of lactation. To study glucose metabolism in peak-, late-, and nonlactating cows, the expression of erythrocyte-type glucose transporter (GLUT1) and the insulin-responsive glucose transporter (GLUT4) in the mammary gland, adipose tissue, and muscle were assessed by Western blotting and real-time PCR. Our results demonstrated that the mammary gland of lactating cows expressed a large amount of GLUT1, whereas the mammary gland of nonlactating cows did not (P < 0.05). On the other hand, adipose tissue of late and nonlactating cows expressed a large amount of GLUT1, whereas the adipose tissue of peak-lactating cows did not (P < 0.05). There were no significant differences in the abundance of GLUT4 mRNA in adipose tissue and muscle, whereas GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was greater (P < 0.05) in nonlactating cows than in peak- and late-lactating cows. The results of the present study indicate that in lactation, GLUT1 expression in the mammary gland and adipose tissue is a major factor for insulin-independent glucose metabolism, and the expression of GLUT4 in muscle and adipose tissue is not an important factor in insulin resistance in lactation; however, the plasma insulin concentration may play a role in insulin-dependent glucose metabolism. Factors other than GLUT4 may be involved in insulin resistance.

Plasma lipid concentrations in preruminant calves fed whole milk with whey protein isolate.

The objective was to investigate the acute effects of retinol acetate added to whey protein isolate (WPI) on postprandial changes in plasma retinol (experiment 1) and the acute effects of milk fat added to WPI on triglyceride (TG), chylomicrons and very low density lipoprotein (VLDL), and fatty acid concentrations (experiment 2) in suckling calves at 1 and 6 wk of age. In experiment 1, 16 Holstein male calves were alloted to 2 equal groups. On the days of measurement, the calves were fed at 0900 h whole milk [4% of body weight (BW)] mixed with vitamin A acetate (500,000 IU) with or without WPI (0.04% of BW). At 1 wk of age, significantly higher postfeeding concentrations of plasma retinol were observed in the calves fed milk with WPI. At 6 wk of age, no differences in the plasma retinol concentrations were observed between 2 groups. On the days of measurement in experiment 2, 16 male calves were fed at 0900 h whole milk (4% of BW) with added milk fat prepared by centrifugation from whole milk (2% of BW) with or without WPI (0.04% of BW). The milk supplemented with fat was prepared on the day before the measurement. At 1 wk of age, significant higher postfeeding concentrations of plasma TG concentrations were obtained in the calves fed WPI than in the control calves, immediately after the meal or from 7 h later onward. Plasma chylomicrons and VLDL concentrations at 1 wk of age were significantly higher in the WPI-fed group than in the control group at 8 h postfeeding. In the calves with the WPI diet, plasma concentrations of myristic, palmitic, stearic, oleic, and linoleic acids at 1 wk of age were significantly higher than those in the control calves at 8 h after feeding. However, chylomicrons and VLDL, and fatty acid concentrations did not differ between the 2 groups after feeding at 6 wk of age. Results indicate that WPI increases plasma lipid concentration of preruminant calves only at 1 wk of age. These data are interpreted to indicate that WPI enhances mainly lipid uptake in the intestines of neonatal calves.

GH secretory responses to ghrelin and GHRH in growing and lactating dairy cattle.

Release of growth hormone (GH) is known to be regulated mainly by GH-releasing hormone (GHRH) and somatostatin (SRIF) secreted from the hypothalamus. A novel peripheral release-regulating hormone, ghrelin, was recently identified. In this study, differences of the GH secretory response to ghrelin and GHRH in growing and lactating dairy cattle were investigated and an alteration of plasma ghrelin levels was observed. The same amounts of ghrelin and GHRH (0.3 nmol/kg) were intravenously injected to suckling and weanling calves, early and mid-lactating cows and non-lactating cows. Plasma ghrelin levels were also determined in dairy cattle in various physiological conditions. The peak values of ghrelin-induced GH secretion were increased in early lactating cows compared to those in non-lactating cows. The relative responsiveness of GH secretion to ghrelin was also increased compared with that to GHRH in early lactating cows. GH secretory responses to GHRH were blunted in mature cows with and without lactation. Conversely, GHRH-induced GH secretory response was greater than that to ghrelin in calves, and also greater in calves than in mature cows. Plasma ghrelin concentrations were elevated in early lactating cows compared to those in non-lactating cows. Plasma GH concentrations were higher in suckling calves and early lactating cows compared with those in non-lactating cows. These results suggest that GHRH is an effective inducer of GH release in growing calves, and that the relative importance of ghrelin in contributing to the rise in plasma GH increases in early lactating cows.

Distribution of apolipoprotein E among lipoprotein fractions in the lactating cow.

Apolipoprotein (apo) E plays a key role in regulating plasma levels of lipoproteins. We investigated the serum apoE concentrations in cows during different lactating stages by ELISA. To confirm the distribution of apoE in lipoprotein fractions, cow plasma was separated by gel filtration, ultracentrifugation and agarose gel electrophoresis. The apoE concentrations during early, mid- and late lactating stages in cows were significantly higher than that during the non-lactating stage. In lactating plasma, apoE eluted in high-density lipoprotein (HDL) fractions separated by gel filtration increased. The portion of this apoE in plasma was 49%. However, when lactating plasma was separated by ultracentrifugation, less then 5% apoE was recovered in the HDL fraction, and more apoE was recovered in the non-lipoprotein fraction (d>1.21 g/ml, 46%). In agarose gel electrophoresis, plasma apoE was found in beta-migrating lipoprotein, but it was not present in alpha-migrating lipoprotein. To purify apoE-containing particles, the HDL fraction separated by gel filtration was pooled and the fraction retained on Heparin-Sepharose chromatography collected. Cholesterol was absent from this fraction. These results suggest that apoE-containing particles, which increased during the lactating stage, were not associated with HDL particles, and that lipid-free forms were included in cow plasma.

DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers.

Death-associated protein kinase is a positive regulator of programmed cell death induced by interferon gamma. To investigate the role of epigenetic inactivation of death-associated protein kinase in gastrointestinal cancer, we examined the methylation status of the 5' CpG island of the death-associated protein kinase gene. Methylation of the 5' CpG island was detected in 3 of 9 colorectal and 3 of 17 gastric cancer cell lines, while among primary tumours, it was detected in 4 of 28 (14%) colorectal and 4 of 27 (15%) gastric cancers. By contrast, methylation of the edge of the CpG island was detected in virtually every sample examined. Death-associated protein kinase expression was diminished in four cell lines that showed dense methylation of the 5' CpG island, and treatment with 5-aza-2'-deoxycitidine, a methyltransferase inhibitor, restored gene expression. Acetylation of histones H3 and H4 in the 5' region of the gene was assessed by chromatin immunoprecipitation and was found to correlate directly with gene expression and inversely with DNA methylation. Thus, aberrant DNA methylation and histone deacetylation of the 5' CpG island, but not the edge of the CpG island, appears to play a key role in silencing death-associated protein kinase expression in gastrointestinal malignancies.

Clinicopathologic and prognostic significance of matrilysin expression at the invasive front in human colorectal cancers.

Matrix metalloproteinases (MMPs) have been implicated in tumor progression. Matrilysin, one of the matrix metalloproteinases, is frequently overexpressed in gastrointestinal cancers. The aim of our study was to assess the validity of matrilysin as a prognostic marker of colorectal cancers. Matrilysin expression was immunohistochemically analyzed using formalin-fixed, paraffin-embedded specimens from 113 colorectal cancer patients who had undergone curative surgery. The lumenal surface of neoplastic glands in the superficial layer was apically stained, while the cytoplasm of cancer cells at the invasive front was diffusely stained for matrilysin. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 47 (42%) cases, were judged as being positive for matrilysin. Matrilysin positivity was significantly correlated with the depth of invasion, lymph node metastasis, lymphatic invasion, advanced Dukes' stage and poor outcome. Patients with matrilysin-positive cancer had a significantly shorter overall survival time than those with matrilysin-negative cancer. For patients with intermediate invasive tumor (T2 or T3), only matrilysin was a significant prognostic variable for predicting overall survival in multivariate analysis. Matrilysin expression at the invasive front could be an important marker, predicting an unfavorable prognosis after surgical treatment in patients with colorectal cancer.

Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads.

Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.

Measurement of circulating biliary glycoprotein (CD66a) in liver diseases.

PURPOSE: Biliary glycoprotein (BGP), a member of the carcinoembryonic antigen (CEA) gene family, is produced by hepatocytes, and is suggested to function as a cell adhesion molecule, mouse hepatitis virus receptor, and tumor suppressor. Our aim was to establish an enzyme immunoassay for circulating BGP and to study its significance in liver diseases. METHODS: For enzyme immunoassay, a monoclonal antibody (mAb), TS135, which recognizes BGP was used as a catcher, and biotin-labeled polyclonal anti-CEA antibodies were used as a tracer. Seventy-six serum specimens obtained from patients with various liver diseases were submitted to the assay. RESULTS: The incidence of positivity for antigen TS135 in the serum samples of the 76 patients was 57.9%. The most significant correlation among conventional liver function tests was found between antigen TS135 and gamma-glutamyl transpeptidase (gamma-GTP). However, among the 56 patients whose serum antigen TS135 and gamma-GTP levels could be measured simultaneously, 5 were antigen TS135-positive and gamma-GTP-negative (8.9%) and 6 were antigen TS135-negative and gamma-GTP-positive (10.7%). The increased serum level of antigen TS135 in 6 cholangiocellular carcinoma (CCC) patients led us to the immunohistochemical study of CCC, in which 8 of the 8 tissue specimens tested were positive for mAb TS135, indicating the production of the antigen from CCCs. CONCLUSIONS: This preliminary study suggests that the circulating antigen TS135 level correlates with gamma-GTP in liver diseases, but that TS135 may also have a unique significance, different from that of gamma-GTP, as a liver function test.

Effects of heat exposure on adrenergic modulation of insulin and glucagon secretion in sheep.

The effects of heat exposure on the adrenergic modulation of pancreatic secretion were investigated. Five ewes fed at maintenance level (ME base) were housed in thermoneutral (TN; 20 degrees C) and hot (30 degrees C) environments. Heat exposure caused an increase in respiration rate and a slightly higher rectal temperature, and decreases in basal insulin and glucose concentrations. Infusions of saline plus epinephrine caused increases in glucagon and glucose concentrations, and no significant change in insulin secretion. Phentolamine (an adrenergic alpha-antagonist) plus epinephrine augmented insulin secretion; however, this insulin secretory response was inhibited by heat exposure. Propranolol (a beta-antagonist) plus epinephrine produced a slight decrease in insulin secretion in the TN environment, whereas no effect was observed during heat exposure. While glucagon secretion through alpha-adrenergic stimulation was not affected by heat exposure, homeostatic signals controlling insulin release seemed to be affected during heat exposure. We thus hypothesised that insulin concentration is decreased in sheep fed at maintenance level in hot environments, and that this response is mediated in part by a modulation of beta-adrenergic function.