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.

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.

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.

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.

[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.

Postprandial changes in plasma concentrations of growth hormone and ghrelin around weaning in the goat.

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.

Effects of long-chain fatty acids on cytosolic triacylglycerol accumulation and lipid droplet formation in primary cultured bovine mammary epithelial cells.

Mammary epithelial cells have recently been shown to express and secrete leptin into milk and to accumulate triacylglycerol (TAG) in cytosol. We examined the effects on the accumulation of cytosolic TAG of free fatty acid addition to the medium bathing bovine mammary epithelial cells (bMEC). Both saturated (palmitic and stearic) and unsaturated (oleic and linoleic) fatty acids stimulated the accumulation of TAG in a concentration-dependent manner from 50 to 400 microM and the expression of mRNA expression for CD36, which is involved in the uptake and secretion of long-chain fatty acids. However, leptin mRNA expression and lipid droplet formation were significantly increased only by the addition of unsaturated, but not saturated, fatty acids. Interestingly, both types of fatty acids stimulated alphas1-casein mRNA expression. These data suggest that the expression of leptin is related to droplet formation, whereas CD36 is related to cytosolic TAG accumulation, and that fatty acids or cytosolic TAG accumulation also have a role to accelerate differentiation of bMEC as shown by casein synthesis.

Suppressing effects of bisphenol A on the secretory function of ovine anterior pituitary cells.

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.

Rapid suppressing action of insulin-like growth factor-I (IGF-I) on GH release from anterior pituitary cells of goats.

Goat anterior pituitary cells were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), insulin, and growth hormone (GH) on basal and GH-releasing hormone (GHRH)-induced GH release. Changes in cellular Ca2+ concentrations were also assessed to enable discussion of the cellular mechanisms of IGF-I. The cells were cultured for 48 h, and then stimulated with GHRH (10 nmol/l) for 30 min, with or without each test substance. In the control cells, IGF-I (10 and 100 ng/ml) significantly raised the basal, but did not change GHRH-induced GH release, resulting in the abolishment of GH release induced by GHRH in the presence of 100 ng/ml IGF-I. However, there was no significant effect of insulin (10, 100, and 1000 microU/ml) on basal and GHRH-induced GH release. In the cells cultured for 48 h with each test substance but stimulated for 30 min without the test substance, no significant change in the basal and GHRH-stimulated GH release was observed. Regardless of treatment, there was no significant effect on intra-cellular GH content. Analysis with a confocal laser microscope revealed that IGF-I (100 ng/ml) significantly increased the basal, but significantly reduced GHRH (10 nmol/l)-induced increase in cellular Ca2+ concentrations. From these findings we conclude that IGF-I exerts an acute suppressing action on the GHRH-induced GH release, which partly involves changes in cellular Ca2+ metabolism in goat somatotrophs.

Existence of GPR40 functioning in a human breast cancer cell line, MCF-7.

GPR40, which has recently been identified as a G-protein-coupled cell-surface receptor for long-chain fatty acids, was assessed in a human breast cancer cell line (MCF-7). We detected GPR40 mRNA by RT-PCR and found that oleate and linoleate, but not palmitate or stearate, caused an increase in cellular Ca(2+) concentrations, which was partially blocked by the pertussis toxin (PTX) treatment. We examined the expression of GPR40 mRNA by quantitative RT-PCR in the relation to cell number. It was significantly increased at the beginning and at the end of cell proliferation. These results indicate the possibility that GPR40 for long-chain fatty acids may be involved in cellular function such as cell proliferation, providing a new perspective for the action of long-chain fatty acids on mammary epithelial cells.

Effects of acetate and butyrate on the expression of leptin and short-form leptin receptor in bovine and rat anterior pituitary cells.

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.

Effects of nutritional conditions around weaning on carbonic anhydrase activity in the parotid gland and ruminal and abomasal epithelia of Holstein calves.

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.

RCAS1 as a tumour progression marker: an independent negative prognostic factor in gallbladder cancer.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis in immune cells bearing the RCAS1 receptor. We sought to determine RCAS1 involvement in the origin and progression of gallbladder cancer, and also implications of RCAS1 for patient survival. RCAS1 expression was examined immunohistochemically in 110 surgically resected gallbladder specimens. The gallbladders represented 20 cases of cholecystitis with no associated pancreaticobiliary maljunction; 23 cases of cholecystitis with pancreaticobiliary maljunction; 14 cases of adenomyomatosis; 7 adenomas; and 46 cancers. High expression of RCAS1 (immunoreactivity in over 25% of cells) was observed in 32 of the 46 cancers (70%), but not in other diseases, including pre-cancerous conditions. RCAS1 immunoreactivity was associated with depth of tumour invasion (P = 0.0180), lymph node metastasis (P = 0.0033), lymphatic involvement (P = 0.0104), venous involvement (P = 0.0224), perineural involvement (P = 0.0351) and stage by the tumour, nodes and metastases (TNM) classification (P = 0.0026). Thus, RCAS1 expression may be a relatively late event in gallbladder carcinogenesis, possibly promoting tumour progression. Cox regression multivariate analysis demonstrated RCAS1 positivity to be an independent negative predictor for survival (P = 0.0337; risk ratio, 12.690; 95% confidence interval, 1.216-132.423). High expression of RCAS1 significantly correlated with tumour progression and predicted poor outcome in gallbladder cancer.

Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts.

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.

Effects of adenosine 5'-triphosphate and growth hormone on cellular H+ transport and calcium ion concentrations in cloned bovine mammary epithelial cells.

The present experiment was carried out to investigate the effects of exogenous adenosine 5'-triphosphate (ATP) and growth hormone (GH) on cellular H(+) efflux rate (extracellular acidification rate) and Ca(2+) concentration ([Ca(2+)](c)) in cloned bovine mammary epithelial cells (bMEC) raised from the mammary gland of a 26-day-pregnant Holstein heifer. Perifusion of 2-day cultured cells with a medium containing ATP (10, 100 and 1000 micromol/l) for 30 min caused a significant and concentration-dependent increase in the cellular H(+) efflux rate. ATP application (100 micromol/l) caused a transient and large increase in [Ca(2+)](c) in all cells. In contrast, perifusion with a medium containing bovine GH at 10, 50 and 250 ng/ml for 30 min caused a significant decrease in the cellular H(+) efflux rate in a concentration-dependent manner. However, bovine GH application (50 ng/ml) caused a small decrease followed by an increase, in some cases, in [Ca(2+)](c). In bMEC treated with lactogenic hormones (1 microgram/l prolactin, 1 nmol/ml dexamethasone and 5 microgram/ml insulin) for 2 days, the increased H(+) efflux rate induced by ATP was significantly reduced, whereas the negative response induced by GH was inversely and significantly changed to the positive. Treatment of the cells with lactogenic hormones reduced the increase in [Ca(2+)](c) induced by ATP stimulation, while it enhanced the increase in [Ca(2+)](c) induced by GH stimulation. Application of ATP or GH did not cause any significant changes in [pH](c). Treatment with lactogenic hormones enhanced GH receptor (GHR) transcription that was determined by RT-PCR. From these results, we conclude that exogenous application of ATP and GH causes prompt and significant responses in H(+) transport and [Ca(2+)](c) that were significantly changed in the opposite direction by the treatment with lactogenic hormones. The lactogenic hormone treatment also enhanced GHR transcription, which may change post-receptor signal transduction systems for both agents in the bMEC.

Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin: their interactions with actin.

This investigation ascertains whether, in (smooth muscle) myosin, certain residues engage in functional interactions with their actin conjugates in an actomyosin complex. Such interactions have been postulated from putting together crystallographic models of the two proteins [Rayment, I., Rypniewski, W. R., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M. (1993) Science 261, 50-58]. Here, in several instances, we ask whether mutation of a particular residue significantly impairs a function, and find that the answers are largely rationalized by the original postulation. Additionally, a novel element emerges from our investigation. To assess function, we test the wild type and mutant systems as they perform in the steady state of ATP degradation. In doing so, we assume, as usual, that degradation proceeds from an early stage in which the complex forms (and is described by parameter K(app)) to a later stage during which the product leaves the complex (and is described by parameter V(max)). Interestingly, certain defects induced by the mutations are associated with changes in K(app), and other defects are associated with changes in V(max), suggesting that our procedure at least roughly distinguishes between events according to the time in the degradation at which they occur. In this framework, we suggest that (1) in the actin-myosin association phase, cationic residues Lys-576 and Lys-578 interact with anionic residues of the so-called second actin, and (2) in the product leaving phase, hydrophobic residues Trp-546, Phe-547, and Pro-548, as well as the Thr-532/Asn-533/Pro-534/Pro-535 sequence, sever connections with the so-called first actin. The role of Glu-473 is also examined.

The significance of thymidine phosphorylase expression in colorectal cancer.

We measured thymidine phosphorylase activity in colorectal cancer tissue and conducted immunostaining to investigate enzyme expression in the tumor tissue. The results showed a correlation between staining ratio of thymidine phosphorylase and cancer progression as well as a correlation between enzyme activity and staining ratios of cancer cells and stromal cells. Since enzyme activity levels can be judged by staining ratios, this method may be useful for assessing cancer malignancy.