Inverse genetics tracing the differentiation pathway of human chondrocytes.

OBJECTIVE: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process. DESIGN: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway. RESULTS: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9. CONCLUSIONS: This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported.

CD204⁺ tumor-associated macrophages are associated with clinical outcome in canine pulmonary adenocarcinoma and transitional cell carcinoma.

Tumor-associated macrophages are abundant infiltrating cells in the tumor microenvironment (TME). Macrophages can be classified into several types of subsets based on their immune responses. Among those subsets, M2 macrophages contribute to anti-inflammatory responses and create an immunosuppressive environment that promotes tumor cell proliferation. In a previous study, human cancer patients with high M2 macrophages showed a worse prognosis for many types of tumors. However, studies examining the relationship between M2 macrophages and clinical outcomes in canine tumors are limited. In the previous human and canine studies, CD204 has been used as the marker for detecting M2 macrophages. Then we evaluated CD204+ and total macrophages infiltration and its association with clinical outcomes in canine solid tumors. In this study, we examined dogs with oral malignant melanoma (OMM), pulmonary adenocarcinoma (PA), hepatocellular carcinoma (HCC), and transitional cell carcinoma (TCC). Compared to healthy tissues, CD204+ and total macrophages were increased in OMM, PA, and TCC, but not in HCC. High CD204+ macrophage levels were significantly associated with lung metastasis in TCC (P = 0.030). Kaplan-Meier analysis revealed that high CD204+ macrophage levels were associated with shorter overall survival (OS) in canine patients with PA (P = 0.012) and TCC (P = 0.0053). These results suggest that CD204+ macrophages contribute to tumor progression and could be a prognostic factor in dogs with PA and TCC.

Duodenal expression of antimicrobial peptides in dogs with idiopathic inflammatory bowel disease and intestinal lymphoma.

Although antimicrobial peptides (AMPs) play an integral role in the regulation of intestinal microbiota and homeostasis, their expression in canine gastrointestinal diseases, including idiopathic inflammatory bowel disease (IBD) and intestinal lymphoma, remains unknown. The objective of this study was to investigate the intestinal expression of AMPs in dogs with IBD or intestinal lymphoma. IBD was diagnosed in 44 dogs, small cell intestinal lymphoma in 25 dogs, and large cell intestinal lymphoma in 19 dogs. Twenty healthy beagles were used as normal controls. Duodenal mRNA expression of six representative AMPs - lactoferrin, lysozyme, cathelicidin, secretory leukocyte peptidase inhibitor (SLPI), bactericidal/permeability increasing protein (BPI), and canine beta defensin (CBD103) - was quantified by real-time reverse transcription polymerase chain reaction. The relative expression of BPI, lactoferrin, and SLPI was significantly higher in dogs with IBD and intestinal lymphomas than in healthy controls. Interestingly, the expression patterns of AMPs differed between dogs with IBD and those with intestinal lymphomas, especially small cell lymphoma. Increased expression of BPI differentiated IBD from dogs with small cell intestinal lymphoma, with a sensitivity of 93.2%, a specificity of 100%, and an area under the curve of 0.955. These results suggest that the expression patterns of AMP aid in the diagnosis of canine IBD and intestinal lymphoma, although it remains uncertain whether the altered AMP expression is the cause or effect of mucosal inflammation.

Expression of apoptosis inhibitor of macrophages in tissue macrophages, leukocytes and vascular endothelial cells of dogs.

Apoptosis inhibitor of macrophages (AIM) is a protein which plays important roles in controlling the immune response and inflammation in human and mice. In dogs, AIM is reported to be expressed in cancerated macrophages and regulate the survival of these tumor cells. In this study, to elucidate the physiological expression pattern of AIM in dogs, systemic expression and distribution of AIM of dogs were investigated. Mature healthy Beagles were used. Various tissues, peripheral blood cells, and bone marrow cells of normal dogs were collected for in situ hybridization, real-time RT-PCR, and immunohistochemistry. AIM mRNA and protein were expressed in tissue macrophages of the spleen, liver, lungs, and lymph nodes, but not in the microglia of the cerebrum. Proximal tubules in the kidney also expressed AIM protein. Monocytes and B lymphocytes in circulating blood and a part of microvasculature endothelial cells showed AIM expression at both the mRNA and protein levels. In the bone marrow, early-stage monocyte progenitor-like cells expressed AIM mRNA and protein. These results clarified that AIM is expressed in more cell types than previously reported in human and mice. These data spread the possibility of AIM physiological functions and implies the relationship of AIM to the maturation of macrophage-strain cells in dogs and other species.

Association of tumour-infiltrating regulatory T cells with adverse outcomes in dogs with malignant tumours.

Regulatory T cells (Tregs) infiltrate into a variety of tumour tissues and associate with poor prognosis in humans. However, data on association of Treg infiltration with prognosis is limited in canine tumours. The purpose of this study was to examine the number of tumour-infiltrating Tregs and its association with overall survival (OS) in dogs with malignant tumours. The following 168 canine tumours were included: 37 oral malignant melanomas (OMMs); 14 oral squamous cell carcinomas (OSCCs); 16 pulmonary adenocarcinomas (PAs); 37 mammary carcinomas (MCs); 36 mast cell tumours (MCTs) and 28 hepatocellular carcinomas (HCCs). Normal tissues were obtained from 8 healthy dogs as controls. The number of forkhead box P3 (Foxp3)-positive Tregs in intratumoral and peritumoral areas was investigated by immunohistochemistry. OS was compared between high and low Treg groups. The number of intratumoral and peritumoral Foxp3-positive Tregs was significantly higher in OMM, OSCC, PA and MC compared with each normal tissue. There were few Foxp3-positive Tregs in MCT and HCC. With intratumoral Tregs, the OS in the high Treg group was significantly shorter than that in the low Treg group in OMM, OSCC and PA. With peritumoral Tregs, there was no significant difference for OS between the 2 groups in each tumour type. These results suggest that Tregs infiltrate into a variety of canine tumours and the abundance of Tregs are associated with poor prognosis in some solid tumour types.

Endoscopic Cytology for the Diagnosis of Chronic Enteritis and Intestinal Lymphoma in Dogs.

Although cytology is a rapid diagnostic procedure in dogs, the cytologic criteria of endoscopic biopsies for chronic enteritis and intestinal lymphoma are not well defined. An immediate diagnosis using cytology would benefit patients by enabling prompt initiation of therapy. The objective of this study was to investigate the correlation between the results of endoscopic cytology and histopathology. In this study, 167 dogs with clinical signs of chronic gastrointestinal disease were included. On the basis of histopathology, the following diagnoses were determined: lymphocytic-plasmacytic enteritis in 93 dogs; eosinophilic enteritis in 5 dogs; small cell intestinal lymphoma in 45 dogs; and large cell intestinal lymphoma in 24 dogs. Two clinical pathologists retrospectively evaluated the endoscopic cytology of squash-smear preparations. The cytologic diagnoses of inflammation, small cell lymphoma, and large cell lymphoma were based on the severity of lymphocyte infiltration, the size of infiltrated lymphocytes, and eosinophil/mast cell infiltration. The clinical severity score was significantly increased along with the degree of lymphocyte infiltration evaluated by cytology. The cytologic diagnosis was in complete agreement with the histopathologic diagnosis in 136 of 167 (81.4%) cases. For the differentiation between enteritis and lymphoma, endoscopic cytology had a sensitivity of 98.6%, a specificity of 73.5%, a positive predictive value of 72.3%, and a negative predictive value of 98.6%. The log-rank test and Cox regression analysis showed that the results of cytology predicted the prognosis. These results suggest that endoscopic cytology is a useful technique to aid diagnosis of intestinal inflammation and lymphoma in dogs.

Geographical variation in the skeletal morphology of red jungle fowl.

1. The skulls and postcranial skeletons of the red jungle fowl (Gallus gallus) were compared osteometrically between the populations from North and South Vietnam, North and Central Laos and Southeast Bangladesh. The populations include the three subspecies of G. g. spadiceus, G. g. gallus and G. g. murghi and were sampled to reveal the geographical morphological variations among populations in G. gallus. 2. The morphometric characteristics of subspecies murghi could be clearly distinguished from those of the other subspecies using a canonical discriminant analysis. However, the size and shape of the skull of the gallus population from South Vietnam were not statistically different from that of the subspecies spadiceus from North Laos. The canonical discriminant scores also clearly indicated that there were morphological similarities in the skulls of the populations from North Laos and South Vietnam. 3. From the results, therefore, it is concluded that red jungle fowls do not exhibit high levels of osteometric variation between geographical localities at least within the Indochinese Peninsula. 4. This contrasts with previous studies which have described these subspecies as having various external morphological differences and have argued that zoogeographical barriers exist between the north and south areas of the Indochinese Peninsula.

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.

Utility of the phylotoxigenic relationships among trichothecene-producing Fusarium species for predicting their mycotoxin-producing potential.

Species of the genus Fusarium are well-known plant pathogens and mycotoxigenic fusaria are associated with health hazards to humans and animals. There is a need to understand the mechanisms of mycotoxin production by Fusarium species and to predict which produce mycotoxins. In this study, the Fusarium phylogenetic tree was first inferred among trichothecene producers and related species. We reconstructed the maximum likelihood (ML) tree based on the combined data from nucleotide sequences of rDNA cluster regions, the ß-tubulin gene (ß-tub) and the elongation factor 1α gene (EF-1α). Second, based on this tree topology, the ancestral states of the producing potential of type A and B trichothecenes (TriA and TriB), zearalenone (ZEN), moniliformin (MON), beauvericin (BEA) and enniatins (ENN) were reconstructed using the maximum parsimony (MP) method based on the observed production by extant species as reported in the literature. Finally, the species having the potential to produce each of these six mycotoxins was predicted on the basis of the parsimonious analysis. The ML tree indicated that the Fusarium species analysed in this study could be divided into two major clades. Clade I was divided into four distinct subclades: I-a, I-b, I-c and I-d. Furthermore, the parsimony reconstruction suggested that the potential for producing MON and ZEN was gained or lost only once, and that the producing potential for TriA and TriB, BEA and ENN was repeatedly gained and lost during the evolutionary history of the Fusarium species analysed in this study. Interestingly, the results showed the possibility that several species, about which reports were scarce with regard to mycotoxin production, have the potential to produce one or more of the six evaluated in this study. The phylogenetic information therefore helps one to predict the mycotoxin-producing potential by Fusarium species, and these "phylotoxigenic relationships" may be useful for predicting the pathogenicity of fungi.

Temperature distributions of electron beam-irradiated samples by scanning electron microscopy.

An electron beam (EB) generated by a scanning electron microscope (SEM) was used to irradiate two samples having different thermal conductivities, and the resulting temperatures of the EB-irradiated areas as well as the temperature distributions within the samples were then measured using a thermal camera. These measurements showed overall increases in sample temperatures, as well as revealed temperature rises at the EB-irradiated areas that had little difference with one of the theoretical predictions. Differences between the actual and the predicted temperature measurements were analysed in terms of the accuracy with which parameters could be estimated. The temperature distributions of the samples were measured and, On the basis of the results, it was hypothesized that the temperature differential over an irradiated sample will be inversely correlated with its thermal conductivity.

Coxa morphologically adapted to large egg in aepyornithid species compared with various palaeognaths.

The coxa of palaeognaths including extinct aepyornithid species was compared by means of osteology, computed tomography (CT) imaging and macroscopic anatomy. The pre-acetabular area of the ilium was proportionally much larger in width and height in aepyornithid species than in other extant palaeognaths. The post-acetabular area of the ilium, ischium and pubis was relatively short and bilaterally extended in aepyornithid species. Although the Aepyornis species might have produced egg with a major axis exceeding 300 mm in major axis, the short post-acetabular coxa and widely opened ischium and pubis would not have been able to stably hold the eggs in the posterior space of the pelvic cavity unlike the situation in the ostrich. As aepyornithid species resembled the kiwi in measurement ratios of length and width in the pre-acetabular area, a functional-morphological model of the coxa in the aepyornithid species can be proposed based on that of the kiwi. From our data, we suggest that the extinct species of Aepyornis and Mullerornis species effectively used the anterior space of the pelvic cavity to support their extraordinary large egg.

Plasma atrial natriuretic peptide in healthy calves and calves with congenital heart disease.

BACKGROUND: The basic and clinical implications of evaluating plasma atrial natriuretic peptide (ANP) concentration in calves are unknown. OBJECTIVE: To investigate the relationship between the plasma ANP concentration and left ventricular end-diastolic pressure (LVEDP) in healthy calves subjected to volume overload (Study 1), and to compare the plasma ANP concentration in calves with or without heart disease (Study 2). ANIMALS: Six healthy calves were used in Study 1; disease calves and sick calves with (n = 9) and without congenital heart disease (CHD) (n = 9) were used in Study 2. METHODS: In Study 1, LVEDP in anesthetized calves was manipulated by IV administration of acetated Ringer's solution (rate of 100 mL/kg/h for 20 minutes) and furosemide. In Study 2, disease calves were identified by blood examination and echocardiography or pathological examination. The plasma ANP concentration was determined by a chemiluminescence enzyme immunoassay for human alpha-ANP. RESULTS: In Study 1, preloading significantly increased the plasma ANP concentration (36 +/- 20-185 +/- 156, P < .01) and LVEDP (-11 +/- 7-2 +/- 12, P < .01) from the baseline. Furthermore, plasma ANP concentrations were strongly correlated with LVEDP (r= 0.61). In Study 2, the plasma ANP concentration was significantly higher in the calves with CHD than in the calves without heart disease (220 [67-970] versus 31 [10-86]; mean [range], P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of plasma ANP concentrations in calves can provide additional information useful for predicting hemodynamic abnormalities.

Evaluation of NT-pro BNP and CT-ANP as markers of concentric hypertrophy in dogs with a model of compensated aortic stenosis.

BACKGROUND: Serum C-terminal atrial natriuretic peptide (CT-ANP) and N-terminal pro B-type natriuretic peptide (NT-pro BNP) concentrations have not been measured serially in dogs with chronic pressure overload of the heart. HYPOTHESIS: We investigated whether serial evaluation of CT-ANP and NT-pro BNP concentrations is a useful guide to the risk of cardiac remodeling in dogs with a model of aortic stenosis. ANIMALS: Six male Beagles. METHODS: After anesthesia, the aorta was constricted with a polyester band and mean left ventricular systolic pressure (LVPs) was 50 mmHg above baseline. Echocardiographic and intracardiac catheter examinations and blood sampling were performed before surgery and 3 and 6 months after surgery. RESULTS: LVP and left ventricular end-diastolic pressure (LVEDP) were significantly higher at 6 months. Compared with baseline, end-diastolic intraventricular septum thickness (IVSd), left ventricular posterior wall thickness (LVPWd), and relative wall thickness (RWT) were significantly increased 3 and 6 months after aortic constriction. Serum CT-ANP concentrations were increased significantly at 3 months and serum NT-pro BNP concentrations were significantly higher 3 and 6 months after aortic constriction. Serum NT-pro BNP concentration was significantly correlated with LVEDP and IVSd whereas serum CT-ANP concentration was not correlated with any measurement. Stepwise regression analysis showed that LVEDP, IVSd, and RWT could predict serum NT-pro BNP. CONCLUSIONS AND CLINICAL IMPORTANCE: This study indicated the differential regulation of NT-pro BNP and CT-ANP concentrations during pressure overload. NT-pro BNP assay may be used as an additional screening method to stratify early-stage ventricular remodeling because of aortic constriction.

Changes in hepatic key enzymes of dairy calves in early weaning production systems.

The objective of the present study was to describe plasma hormonal and metabolite profile and mRNA expression levels and activities of the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and acetyl-coenzyme A (CoA) carboxylase in the liver of male Holstein calves before (1 and 3 wk of age) and after (8, 13, and 19 wk of age) weaning at 6 wk of age. The mean plasma concentration of acetate and beta-hydroxybutyrate increased, and that of plasma lactate and nonesterified fatty acids decreased with week, particularly after weaning. Plasma glucose concentration was lowest at 8 wk of age. The mean plasma concentration of insulin and glucagon did not change with time, and that of cortisol was greatest at 1 wk of age. In the liver, enzyme activity of PC was greatest at 1 wk of age and decreased with time. There was a significant relationship between the activity and the mRNA level for PC. Activity of PEPCK also decreased with week. Acetyl-CoA carboxylase activity tended to decrease with week, and activity at 13 wk of age was lower than that at other times. Expression of PC mRNA, but not that of PEPCK and acetyl-CoA carboxylase alpha, decreased with week. We conclude that the hepatic gluconeogenic enzymes and acetyl-CoA carboxylase activities tend to decrease with age, reflecting changes in plasma metabolites in early weaning production systems.

Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves.

Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, alpha-amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

The semifossorial function of the forelimb in the common rice tenrec (Oryzorictes hova) and the streaked tenrec (Hemicentetes hemispinosus).

The forelimb muscles of the two semifossorial species of Tenrecidae (Oryzoryctinae: common rice tenrec; and Tenrecinae: streaked tenrec) were compared macroscopically with those of the unspecialized terrestrial-arboreal species, the Talazac long-tailed tenrec. The structure of the hand was also observed using three-dimensional reconstructed images from computed tomography data. The two semifossorial species had similar muscle weight ratios in the lateral and long heads of M. triceps brachii and M. teres major. A similar hand skeleton structure (in which the second, third and fourth metacarpals and phalanges act as a digging apparatus) was observed in both species. Our observations confirm that both these species have muscular-skeletal adaptations supporting fossorial locomotion. As each species belongs to a monophyletic subfamily within the Tenrecidae isolated in Madagascar, such semifossorial adaptations are assumed to have evolved convergently.

Suppressing actions of butyrate on growth hormone (GH) secretion induced by GH-releasing hormone in rat anterior pituitary cells.

We assessed the inhibitory effects of butyrate on the growth hormone (GH) secretion in order to investigate the cellular mechanisms in rat somatotrophs. Isolated anterior pituitary cells were cultured in DMEM for several hours, either in the presence (1, 3, or 10mM) or absence of butyrate, and then stimulated with 10(-7)M GHRH for 30 min, in the presence of butyrate at the concentrations used for the previous culture. The increase in GHRH-induced GH release was significantly reduced in a time-dependent and concentration-dependent manner in the cells previously cultured with butyrate. GH content (the sum of GH released into the medium induced by GHRH stimulation and the GH remaining in the cells after stimulation) was reduced by the culture of cells in the presence of butyrate, which was also inversely dependent on the concentrations used for the culture. Simultaneous addition of an L-type Ca(2+) channel blocker, nifedipine (10 pM), to the medium during 10(-9)M GHRH stimulation significantly reduced the stimulated GH release, which was further significantly decreased by a simultaneous addition of 10 mM butyrate. Butyrate blunted the GHRH (10(-9)M)-induced increase in cellular cyclic AMP and calcium ion concentrations, the activity of protein kinases (A and C), and GHmRNA expression. The expression of mRNA for GPR 41 and 43, known as receptors for short-chain fatty acids, was confirmed in the anterior pituitary cells. These findings suggest that butyrate inhibits GHRH-induced GH release as well as GH production, and the cellular inhibitory actions of butyrate occur in diverse cellular signaling pathways of rat somatotrophs.

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.

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.

Correlation between spontaneous feeding behavior and neuropeptide Y profile in the third ventricular cerebrospinal fluid of goats.

Feeding behavior is regulated by neural signals in the hypothalamus, but secretory activities of these signals in vivo and their relationship with spontaneous feeding remain to be solved. In the present study, we investigated the correlation between neuropeptide Y (NPY) and somatostatin (SRIF) profiles in cerebrospinal fluid (CSF) and spontaneous feeding behavior in goats. CSF samples were collected every 15 min for 8 h from the third ventricle and feeding behavior was observed throughout the experimental period. The spontaneous feeding behavior, the mean duration of which was 58 min, occurred with an interval of 146 min. NPY in the CSF fluctuated in an episodic fashion with a 145 min interval. Each NPY episode was followed by spontaneous feeding with a time lag of 24 min. SRIF levels in CSF changed more frequently in a pulsatile manner and were related to neither NPY profiles nor feeding behavior. These results suggest that NPY, but not SRIF, is a physiological signal to drive feeding in goats.