Toshiyuki Saito

Genomic structure of swine taste receptor family 1 member 3, TAS1R3, and its expression in tissues.

Taste receptor family 1 member 3, TAS1R3, is shown to be involved in sweet and umami tastes in mouse, and the nucleotide sequence of the gene has been reported in rat, gorilla, and human. Pigs are frequently used as models for human diseases, and are also considered to be source animals for xenotransplantation to humans due to their anatomical and physiological similarities to humans. Therefore, in the present study, the genomic structure of the swine TAS1R3 gene was determined, and TAS1R3 expression was studied in various swine tissues. The gene was shown to reside on swine chromosome 6q22→q23, from which three types of mRNAs were generated: 3,752 bp derived from six exons in tongue, 3,704 bp from six exons and 3,630 bp from seven exons in testis. The 6 exons/5 introns were structurally similar to those of humans and mice, but the 7 exons/6 introns structure of TAS1R3 was first observed in swine. High expressions of TAS1R3 were revealed in tongue, kidney, and testis by real-time PCR. The expression profile of the tissues except for kidney was similar to that of mouse. When in situ hybridization using an RNA probe for TAS1R3 was performed on swine tongue and testis tissues, TAS1R3 expressions were revealed in tongue circumvallate papillae, fungiform papillae, mucosal epithelium, follicular B lymphocytes, lymphocytes in submucosal tissues of lingual tonsil, and spermatogenic cells. Using peripheral mature B lymphocytes, the expression of TAS1R3 in B lymphocytes was further confirmed by real-time PCR and sequencing of the real-time PCR product.

Platelets prevent acute liver damage after extended hepatectomy in pigs

Background/PurposePlatelets develop tissue repair and promote liver regeneration. We investigated whether platelets prevented acute liver damage after extended hepatectomy in pigs.MethodsThrombocytosis was induced by the following two methods; afterwards 80% hepatectomy was performed in pigs. In the first method, the pigs received administration of thrombopoietin [TPO (+) group], and they were compared with a control group [TPO (−) group]. In the second method, the pigs received a splenectomy [Sp (+) group], and theywere compared with another control group [Sp (−) group]. Platelet counts, biochemical examination of blood, and histopathological findings of the residual liver were examined.ResultsSerum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and total bilirubin (T-Bil) levels were significantly decreased in the thrombocytotic groups compared with the control groups in the early period after hepatectomy. In the histopathological findings, hemorrhagic necrosis with a bile plug was observed in the control groups, but this phenomenon was not observed in the thrombocytotic groups. On transmission electron microscopy, the sinusoidal endothelial lining was destroyed and detached into the sinusoidal space with enlargement of Disse’s spaces in the thrombocytotic groups, but these findings were not observed in the control groups.ConclusionAn increased number of platelets prevents acute liver damage after extended hepatectomy.

Nitrogen transformation in four Japanese soils following manure+urea amendment

Understanding the effects of manure amendment on soil nitrogen (N) mineralization is crucial for a better assessment of N supplying potential of manure to maximize crop yield and minimize possible contamination of surface and belowground water resources. The effects of manure amendment on N mineralization were therefore studied under laboratory conditions. Surface (0–30 cm) samples of four Japanese soils: Andosol, red-yellow soil, masatsuchi, and sandune soil were each treated with chicken manure (PM), cattle manure (CM), swine manure (SM), ostrich manure (OM), and urea, and incubated for 8 weeks at 25°C. Nitrogen mineralization was studied in a 2 M KCl extract of samples collected at weekly intervals. The results showed that N mineralization was affected by both soil and manure types. NH4-N was found to be the dominant form of mineral N while appreciable increases in NO3 levels were observed during the 7th week. The rate of net N mineralization was highest in urea-treated soils, and the stimulating effect of urea on soil N mineralization was noted in the soils. Nitrogen mineralization from manure-treated soils was very low, suggesting that their N effect in the short-run should not be overvalued. In Andosol, CM and OM treatments exhibited negative N fertilizer effects. Ostrich and chicken manures showed high potentials for raising the pH of masatsuchi and sandune soils.

Determination of stereotaxic coordinates for the hippocampus in the domestic pig

In this study a stereotaxic instrument and a stereotaxic procedure based on external skull structures, to be used in prepubertal male Landrace pigs weighing less than 30 kg, is described. The instrument represents an adaptation of the apparatus designed by Marcilloux et al., Brain Res Bull 1989;22:591-597, but we have modified the instrument for stereotaxic procedures based on external skull structures, instead of intracerebral structures necessitating ventriculography (Marcilloux et al., Brain Res Bull 1989;22:591-597). For this reason the U-shaped frame and the ear-bar supports have been changed allowing the three-dimensional placement of the ear-bars into the oblique auditory canals. Firm fixation of the skulls of pigs weighing less than 30 kg, was furthermore secured with modified infraorbital ridges and hard palate pieces. Measurements of distances between external skull structures in animals of the same sex, age and weight showed a negligible variation, thus enabling definition of the horizontal, frontal and sagittal zero planes using external skull structures alone. Stereotaxic coordinates for the hippocampal region of male Landrace pigs weighing 10 kg were then provided and the coordinates from two different levels of the hippocampal region are presented in the text. The reliability of the stereotaxic instrument was finally secured by intrahippocampal injections of ink at predetermined coordinates.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.

Development of a stereotaxic instrument for study of the bovine central nervous system

We have developed a bovine stereotaxic instrument in this study to be used for Holstein cattle from weanling to adult age. The bovine stereotaxic instrument is derived from the Horsley-Clarke principle. A pair of straight ear-bars and orbital ridge pieces with their carriers, head clamps, AP bars are symmetrically arranged across the head pace. For holding the heavy heads, a hard palate and a mandibular support are designed to be placed on the surface of the base frame. A new manipulator with long working distance has also been made. For ventriculography, a holder inserting an X-ray film cassette is placed just next to the AP bar. Finally, the instrument was combined together with an oil-operated lift, so that it can be adjusted to a proper height. Details of this instrument are described in this paper.

Radiotelemetry recording of electroencephalogram in piglets during rest

A wireless recording system was developed to study the electroencephalogram (EEG) in unrestrained, male Landrace piglets. Under general anesthesia, ball-tipped silver/silver chloride electrodes for EEG recording were implanted onto the dura matter of the parietal and frontal cortex of the piglets. A pair of miniature preamplifiers and transmitters was then mounted on the surface of the skull. To examine whether other bioelectrical activities interfere with the EEG measurements, an electrocardiogram (ECG) or electromyogram (EMG) of the neck was simultaneously recorded with the EEG. Next, wire electrodes for recording movement of the eyelid were implanted with EEG electrodes, and EEG and eyelid movements were simultaneously measured. Power spectral analysis using a Fast Fourier Transformation (FFT) algorithm indicates that EEG was successfully recorded in unrestrained piglets, at rest, during the daytime in the absence of interference from ECG, EMG or eyelid movements. These data indicate the feasibility of using our radiotelemetry system for measurement of EEG under these conditions.

A technique for long-term implantation of a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs based on ventriculography

The objectives of this study were to implant a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs, and to maintain the microcatheter for a long time without causing stress. Fourteen pigs (45-60 kg BW) were used. Each pig was anesthetized and the head was orientated on the stereotaxic apparatus. A radiopaque dye was placed into the ventricle via a guide cannula inserted 3.5 mm forward of the bregma. A microcatheter was inserted into the third ventricle using ventriculography, and fixed with dental cement to a metal-mesh protector and screw anchors embedded into the skull. The opposite end of the microcatheter was externalized from the dorsal neck so that corticotropin-releasing hormone (CRH) could be injected easily. Simultaneously, a catheter was fitted in the jugular vein, and the free end of the catheter was externalized from the dorsal neck. Microcatheter-implanted pigs showed a normal progesterone concentration profile, and a constant cortisol level during at least two estrous cycles. Furthermore, intracerebroventricular injections of CRH (25 microg/500 microl) resulted in an increased plasma cortisol concentration (P < 0.05). Thus, the technique developed in this study enables us to approach the third ventricle in post-pubertal freely-moving pigs effectively over a long time, without causing stress.

T-type Ca2+ channels contribute to IBMX/forskolin-and K+-induced Ca2+ transients in porcine olfactory receptor neurons

T-type Ca(2+) channels are low-voltage-activated Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarization above resting potentials. Using Ca(2+) imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca(2+) channels in IBMX/forskolin- and sparingly elevated extracellular K(+)-induced Ca(2+) transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10microM) or Ni(2+) (100microM), the selective T-type Ca(2+) channel inhibitors, IBMX/forskolin-induced Ca(2+) transients in the soma were either strongly (>60%) inhibited or abolished completely. However, the Ca(2+) transients in the knob were only partially (<60%) inhibited. Ca(2+) transients induced by 30mM K(+) were also partially ( approximately 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5mM increase in the extracellular K(+) concentration (7.5mM K(+)), and such responses were completely inhibited by mibefradil or Ni(2+). These results reveal functional expression of T-type Ca(2+) channels in porcine ORNs, and suggest a role for these channels in the spread Ca(2+) transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.

Foetal ECG extraction from maternal body surface measurement using independent component analysis

A method applying independent component analysis (ICA) to detect the electrocardiogram of a prenatal cattle foetus is described. Three channels of signal, one is from chest lead and two are from abdominal leads, are picked up noninvasively by attaching disposable cutaneous electrodes on the body surface of a maternal cow. Measured signals are the mixtures of components including maternal ECG, foetal ECG and random noise. Such mixture procedure is expressed by a time-invariant linear model. To separate foetal ECG from maternal ECG and random noise, the ICA method is applied to rind an optimal separating matrix only using measured signals. The optimization uses hyperbolic tangent as a contrast function and minimizes it based upon the principle of mutual information. The method was examined by simulation signals generated by random mixture of two-signal and one-noise sources. Real signals, measured from a pregnant cow having 177-day gestation, are used to verify the separation. The results show that in simulation, both signal and one noise are clearly separated. Real measurement is successfully distinguished into three signal sources, maternal ECG, foetal ECG and random noise. The results suggested the effectiveness of ICA approach in detecting foetal ECG from maternal body surface measurement.