Atsushi Onuki

Nitric oxide induces vascular endothelial growth factor expression in the rat placenta in vivo and in vitro.

We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

Innervation of Sonic Muscles in Teleosts: Occipital vs. Spinal Nerves

The innervation of sonic muscles in teleosts has been categorized into three types: occipital nerve, spinal nerve, and a combination of occipital and spinal nerves. The innervation patterns of sonic muscles were examined (or re-examined) in seven sonic fish species (rockfish, pinecone fish, sweeper, tigerfish, piranha, dory, and pollack) that use the sonic muscles to vibrate the swimbladder. The peripheral nerves (occipital or spinal) were identified based on skeletal preparations. The sonic muscle innervation was of the occipital type in four species (rockfish, pinecone fish, sweeper, and tigerfish) and of the spinal type in three species (piranha, dory, and pollack); none of the seven species examined showed the combination type. Therefore, we hypothesized that innervation patterns could be divided simply into occipital and spinal types. Moreover, the present results revealed that previously reported innervation patterns are inaccurate for three species (tigerfish, piranha, and dory) re-examined in this study. Therefore, it is important to define the peripheral nerves precisely, by using skeletal preparations, in future investigations of sonic muscle innervation.

Sound characteristics and the sound producing system in silver sweeper, Pempheris schwenkii (Perciformes: Pempheridae)

The sound characteristics and the sound producing system in silver sweeper, Pempheris schwenkii, are described. The main frequency component of the emitted sounds was 100-300 Hz, the mean duration of the sounds being 56 ms. The sounds were produced by paired extrinsic sonic muscles vibrating the swim-bladder. The sonic muscles originated on the pterotic bones and extended to the anterodorsal surface of the swimbladder, and were innervated by two branches of the occipital nerves. Moreover significant sexual dimorphism of the sonic muscles was observed, those in males being three times as large as in females.

Spinal Nerve Innervation to the Sonic Muscle and Sonic Motor Nucleus in Red Piranha, Pygocentrus nattereri (Characiformes, Ostariophysi)

The red piranha, Pygocentrus nattereri, produces sounds by rapid contractions of a pair of extrinsic sonic muscles. The detailed innervation pattern of the sonic muscle of the red piranha was investigated. The sonic muscle is innervated by branches (sonic branches) of the third (S3so), fourth (S4so), and fifth (S5so) spinal nerves. The average total number of nerve fibers contained in the right sonic branches (n = 5; standard length, SL, 71–85 mm) was 151.8 (standard deviation, SD, 28.3). The occipital nerve did not innervate the sonic muscle. The sonic motor nucleus (SMN) in the piranha was identified by tracer methods using wheat germ agglutinin-conjugated horseradish peroxidase; labeled sonic motor neurons were only observed on the side ipsilateral to the sonic muscle injected with the tracer. In the transverse sections, the labeled sonic motor neurons were located in the dorsal zone (mainly large and medium neurons) and in the ventral zone (mainly small neurons) of the ventral horn. In the horizontal sections, the labeled neurons formed a rostrocaudally elongated SMN from the level of the caudal part of the second spinal nerve root to the intermediate region between the fifth and sixth spinal nerve roots. The average number of the labeled neurons (n = 5; SL, 64–87 mm) was 152.6 (SD, 7.3). We conclude that the sonic muscles of the piranha are innervated by approximately 300 sonic motor neurons located only in the spinal cord.

Valproic acid, a histone deacetylase inhibitor, decreases proliferation of and induces specific neurogenic differentiation of canine adipose tissue-derived stem cells.

ABSTRACT Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.

Spinal Nerve innervation to the Sonic Muscle in Walleye Pollack, Theragra Chalcogramma (gadidae: Gadiformes)

Abstract The Walleye Pollack (Theragra chalcogramma) produces sounds by contractions of a pair of intrinsic sonic muscles located on the swimbladder. The innervation pattern of this muscle was investigated. It is innervated by branches (sonic branches) of the third to eighth spinal nerves. The Walleye Pollack was found to be one of the rare sonic fishes with the sonic muscle innervated only by the spinal nerves.

Sound Characteristics and Sonic Motor System in the Pineconefish, Monocentris japonica (Beryciformes: Monocentridae)

Abstract We investigated sound production and the sonic motor system in the Pineconefish, Monocentris japonica. The hand-held Pineconefish produced one type of sound (main frequencies: 100–600 Hz) in both water and air. The sonic organ consists of the swimbladder and a pair of extrinsic sonic muscles originating from exoccipital of the cranium and supracleithrum and inserting on the anterodorsal surface of the swimbladder and partly on vertebrae. The sonic muscle is innervated by occipital nerve branches, not the spinal nerves. Injections of wheat germ agglutinin-conjugated horseradish peroxidase into the sonic muscle resulted in labeled sonic motor neurons ipsilateral to the injections. The labeled neurons were located in the ventral zone of the caudal medulla oblongata, forming a column of sonic motor neurons (or the sonic motor nucleus) from the level of the vagal nerve root to the rostral part of the third ventral root of the occipital nerve. The total number of motor nerve fibers contained in the right and left sonic nerve branches (standard length  =  96 mm) were 363 and 369, respectively. We conclude that the sonic muscles of Monocentris japonica are innervated by approximately 700 sonic motor neurons located only in the caudal medulla oblongata.

Aqueous extracts of Rhizopus oryzae induced nitric oxide production in rat hepatocyte cell line RLN-10

Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22 phox and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.