平尾 温司

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, alpha-smooth-muscle actin (alpha-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor alpha (ER alpha). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERa was not found in the fetal mammary gland, but adipocytes showed the presence of ERa. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Domestic pigs possess a well-developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP-expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP-expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP-expressing cells had longitudinal dendrites. A few GFP-expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP-expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns.

Mammalian tribbles homolog 1 (TRIB1) regulates hepatic lipogenesis and is genetically associated with plasma triglyceride (TG) levels and cholesterol, but the molecular mechanisms remain obscure. We explored these mechanisms in mouse livers transfected with a TRIB1 overexpression, a shRNA template or a control (LacZ) adenovirus vector. The overexpression of TRIB1 reduced, whereas induction of the shRNA template increased, plasma glucose, TG, and cholesterol and simultaneously hepatic TG and glycogen levels. The involvement of TRIB1 in hepatic lipid accumulation was supported by the findings of a human SNP association study. A TRIB1 SNP, rs6982502, was identified in an enhancer sequence, modulated enhancer activity in reporter gene assays, and was significantly (P = 9.39 x 10(-7)) associated with ultrasonographically diagnosed nonalcoholic fatty liver disease in a population of 5570 individuals. Transcriptome analyses of mouse livers revealed significant modulation of the gene sets involved in glycogenolysis and lipogenesis. Enforced TRIB1 expression abolished CCAAT/enhancer binding protein A (CEBPA), CEBPB, and MLXIPL proteins, whereas knockdown increased the protein level. Levels of TRIB1 expression simultaneously affected MKK4 (MAP2K4), MEK1 (MAP2K1), and ERK1/2 (MAPK1/3) protein levels and the phosphorylation of JNK, but not of ERK1/2. Pull-down and mammalian two-hybrid analyses revealed novel molecular interaction between TRIB1 and a hepatic lipogenic master regulator, MLXIPL. Co-expression of TRIB1 and CEBPA or MLXIPL reduced their protein levels and proteasome inhibitors attenuated the reduction. These data suggested that the modulation of TRIB1 expression affects hepatic lipogenesis and glycogenesis through multiple molecular interactions.

AMPA receptor (AMPA-R) complexes consist of channel-forming subunits, GluA1-4, and auxiliary proteins, including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. The combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors, we compared the proteins that copurify with each receptor type in the rat brain. This interactome study identified the majority of known interacting proteins and, more importantly, provides candidates for additional studies. We validate the claudin homolog GSG1L as a newly identified binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes, and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function.

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation. (C) 2009 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3241985]

Previously it has been shown that androgen suppresses transportation-induced increases in plasma adrenocorticotropic hormone (ACTH), possibly by suppressing the secretion of corticotrophin releasing hormone (CRH) or arginine vasopressin (AVP) from the hypothalamus, or secretion of ACTH from the pituitary gland. The aim of the present study was to examine androgen target sites in the caprine diencephalon and pituitary gland using immunohistochemical methods. The androgen receptor (AR) was expressed strongly in the bed nucleus of the stria terminalis, the medial preoptic area, the arcuate nucleus, the ventromedial hypothalamic nucleus and the suprachiasmatic nucleus in the diencephalon. Between 8%, and 11% of CRH and AVP neurons in the paraventricular hypothalamic nucleus (PVN) expressed AR. In the pituitary gland, 7.1%, of corticotrophs expressed AR. The results are consistent with the proposal that androgen acts directly and indirectly on CRH and/or AVP neurons in the PVN. The possibility of a direct action of androgen oil the corticotrophs in the pituitary gland was also considered. (c) 2008 Elsevier Ltd. All rights reserved.

Recent studies have indicated that avian social behavior is influenced by olfactory cues. During the reproductive season a change in the chemical composition of uropygial gland secretion has been reported in some species and the hypothesis that olfactory signals may be produced by this gland has been proposed. To examine this hypothesis we performed two behavioral experiments to determine whether a female's uropygial gland produce chemical signals that Stimulate mating behaviors in domestic chickens. In Experiment 1 the role of the female's uropygial gland in male mating behavior was examined by removing and examining the female's uropygial gland. The frequency of mounts and copulations of intact male birds with sham-operated female birds was significantly higher than with uropygial glandectomized female birds. With respect to the number of waltzing that is one of the courtship displays intact males showed no significant difference between sham-operated female birds and uropygial glandectomized female birds. In Experiment 2 the relationship between male olfaction and the female's uropygial gland was investigated using olfactory bulbectomized male birds. The number of mounts and copulations of sham-operated male birds with sham-operated female bird was significantly higher than with uropygial glandectomized female birds. In contrast olfactory bulbectomized male birds showed no significant differences in the number of mounts and copulations between sham-operated female birds and uropygial glandectomized female birds. These results indicate that intact and sham-operated male birds prefer to mate with female birds with the uropygial gland. The number of courtship waltzing of sham-operated male birds showed no significant difference. However olfactory bulbectomized male birds significantly courted to uropygial glandectomized female birds. Summarizing our results show that while anosmic males did not have any preference both intact and sham-operated male birds chose to mate with female birds having an intact uropygial gland suggesting that mate preference involves in male olfaction and that the female's uropygial gland acts as a source of social odor Cues in domestic chickens. (C) 2008 Elsevier B.V. All rights reserved.

We histologically examined lectin binding patterns in the olfactory bulb of mallard ducks (Anas platyrhynchos) using 21 biotinylated lectins. Positive staining for the N-acetylglucosamine-specific lectins (Bandeiraea simplicifolia II, Datura stramonium, Lycopersicon esculentum, Solanum tuberosum, Triticum vulgare), galactose or N-acetylgalactosamine-specific lectins (Artocarpus intergrifolia, Phaseolus vulgaris erythroagglutinin, Phaseolus vulgaris leucoagglutinin, Ricus communis) and the mannose-specific lectins (Lens culinaris and Pisum sativum) was observed in the olfactory nerve and glomerular layers. Canavalia ensiformis staining showed a similar pattern to that obtained with the lectins and there was also faint staining in the mitral cells. Olfactory nerve axons terminate in the glomeruli, where they make excitatory synapses with the dendrites of mitral cells. This finding indicates that glycoconjugates that bind Canavalia ensiformis play an important role in formation of glomeruli. No positive staining for the other lectins was seen in the olfactory bulb. Based on these results, we conclude that cell surface sugar moieties of the olfactory bulb in mallard ducks express N-acetylglucosamine and mannose residues rather than N-acetylgalactosamine residues. The carbohydrate composition of mallard duck olfactory bulb differed from that of other vertebrates found in previous studies.

A transient receptor potential (TRP) family, TRPV4, is a calcium-permeable swell-activated channel, playing a role in cutaneous mechanosensation. To elucidate the localization in the mechanosensitive endings, we found with immunohistochemistry in mice that TRPV4 was expressed both by small (low threshold) and large (high threshold) dorsal root ganglia neurons. In addition to free nerve endings, TRPV4 was specifically located at cutaneous mechanosensory terminals co-localized with neurofilament 200, including Meissner, Merkel, penicillate and intraepidermal terminals but not including hair follicle palisades. The distribution suggests that the sensation of pressure by mechanosensitive TRPV4 channel is transmitted through A- as well as C-fiber. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

The molecular mechanism of the transmission of changes in the shape of the cell surface to ion channels remains obscure. Ca2+ influx induced by cell deformity is inhibited by actin-freezing reagents, suggesting that the actin microfilament couples with an ion channel. Transient receptor potential vanilloid 4 (TRPV4) is a candidate in the calcium-permeable, swelling-activated mechanosensitive channel in heterogeneously expressed cells. To investigate the mechanosensitive molecular complex, we found that microtubule-associated protein 7 (MAP7) is the mouse TRPV4 C-terminal binding protein. MAP7 was coimmunoprecipitated with TRPV4. The results of a pull-down assay demonstrated that the alignment of amino acids 798-809 of TRPV4 was important in this interaction. TRPV4 and MAP7 colocalized in the lung and kidney. The coexpression of these two molecules resulted in the redistribution of TRPV4 toward the membrane and increased its functional expression. The alignment of amino acids 798-809 of TRPV4 was also important in the functional expression. The activated current was abolished by actin-freezing but not by microtubule-freezing reagents. We therefore believe that MAP7 may enhance the membrane expression of TRPV4 and possibly link cytoskeletal microfilaments.

AQP10 is the newest member of aquaporins in mammals and expressed selectively in the duodenum and the jejunum in human functioning as aquaglyceroporin. Here we report the cloning of the mouse AQP10 gene. The gene is composed of six exons and spans 5.2 kb. The arrangement of the exons is well conserved between mouse and human. However, the initiator methionine is lost because of the mutation at the translation-initiation site. An insertion of four thymine residues in exon 2 and a deletion of a cytosine residue in exon 5 shift the reading frame. Moreover, aberrant exon/intron junction sequences of introns 2, 3, and 4 also shift the reading frame between exons. Genomic Southern blot revealed the mouse AQP10 gene as a single copy gene. The results indicate that the mouse AQP10 gene is a pseudogene. Furthermore, the mouse AQP10 transcript was not detected in the jejunum where the human AQP10 is strongly expressed. (C) 2002 Elsevier Science (USA). All rights reserved.

In order to clarify olfactory system of birds, the efferent and afferent connections of chicken olfactory bulb were investigated using with biocytin and horseradish peroxidase (HRP) methods. From the results of biocytin method, the efferent pathways of chicken olfactory bulb were classified into route 1-3.Route 1 projected to the ipsilateral hyperstriatum accessorium (HA), and hippocampus (Hp). Route 2 had the projections to ipsilateral hyperstriatum intercalatum supremum, hyperstriatum ventrale (HV), neostriatum (N), and nucleus basalis (Bas). Route 3 projected to the bilateral lobus parolfactorius, tuberculum olfactorium tractus fronto-archistriaticus, paleostriatum augmentatum, archistriatum anterior, cortex piriformis, area temporo-parieto-occipitalis, nucleus taeniae, contralateral HA, Hp, HV, N, and Bas. From the results of HRP method, the chicken olfactory bulb received the projections from nucleus septalis medialis, nucleus septalis lateralis, and Hp. In addition, it was appeared that the chicken olfactory bulb has the reciprocal connection with Hp.

The efferent and afferent connections of the bulbus olfactorium (BO) in chickens (Gallus gallus domesticus) were studied using the biocytin and horseradish peroxidase (HRP) methods. Twelve and seven fowls were used for biocytin and HRP studies, respectively. After biocytin was injected into the unilateral BO, many labeled fibers and terminal grains were seen in the bilateral tuberclum olfactorium. Labeled fibers and terminal grains were also found in the lobes parolfactorius, nucleus basalis, neostriatum (N), hyperstriatum accessorium (HA), paleostriatum augumentum (PA), archistriatum pars ventralis (AV), nucleus taeniae, cortex piriformis (CPi) and nucleus septomesencephalici (nSMT), bilaterally. After HRP was injected into the unilateral BO, retrogradely labeled neurons were found bilaterally in the HA as well as the nucleus septalis lateralis and medialis. These results show that the projections of a chicken BO in regard to CPi, Av, and TO are similar to projections of a mammalian's olfactory bulb. A projection from BO to nSMT is similar to that of a reptlian. In addition, projections of BO in regard to the PA, and projections f S to BO in the chicken have not been reported for other vertebrates.