Tatsuo Kubo

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light.

Abstract: Serotonin N‐acetyltransferase [arylalkylamine N‐acetyltransferase (AANAT); EC2.3.1.87] is the rate‐limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT‐PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro‐lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light‐exposure for 2 hr just after lights‐off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%

regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica) : II. Effect of vitamin A deficiency

Abstract: Levels of serotonin N‐acetyltransferase [arylalkylamine N‐acetyltransferase (AANAT): EC2.3.1.87] mRNA, AANAT activity, and melatonin display a rhythmic pattern in both the pineal gland and the retina. It has been shown that vitamin A is required to maintain the rhythm of melatonin synthesis in the pineal gland of Japanese quail. To understand the mechanism underlying the direct relationship among these factors, we developed an assay system sensitive enough to determine AANAT mRNA, AANAT activity, and melatonin content from a single pineal gland of Japanese quail. Positive direct relationships were found among these three parameters. We next deprived Japanese quail of vitamin A by feeding them a vitamin A‐free diet supplemented with retinoic acid, and examined the effects of vitamin A deficiency on the expression of AANAT mRNA in the pineal gland and the retina. Vitamin A deficiency reduced both the expression of AANAT mRNA and melatonin content in the pineal gland. Retinal AANAT mRNA rhythm disappeared in vitamin A‐deficient quails. Moreover, the responsiveness of the pineal gland and the retina to light was reduced by vitamin A deficiency when compared with the control group.

Cloning of Complementary Deoxyribonucleic Acids Encoding Quail (Coturnix coturnix japonica) Retinoic Acid Receptor β Isoforms and Changes in Their Gene Expression During Gonadotropic Growth

Abstract Retinoids have important effects on the development of the reproductive system, where they act via their specific nuclear receptors: retinoic acid receptors (RARα, β, γ) and retinoid X receptors (RXRα, β, γ). The research reported here was conducted in an effort to clone quail RARβ cDNA (qRARβ) and to evaluate the expression of qRARβ mRNAs in different tissues and during the development of gonadotropic organs. Two complete cDNAs of qRARβ1 and qRARβ2 were isolated by a combination of reverse transcription-polymerase chain reaction and 5′- and 3′-rapid amplification of cDNA ends techniques. An RNase protection assay revealed the widespread expression of qRARβ1 and β2 with large tissue-specific variations. The qRARβ1 isoform was predominant in the testis, whereas qRARβ2 was dominant in the other tissues examined with the exception of the brain, where both isoforms were almost equally expressed. In the developing testes, the qRARβ1 mRNA level was high between 30 and 40 days of age, the period during which the testes grew rapidly. The level declined thereafter to its initial level. In contrast, qRARβ2 mRNA did not exhibit obvious changes. In the developing oviducts, both qRARβ1 and β2 mRNAs reached their peak levels by 30 days of age, just before the rapid development of the oviduct occurred, and then decreased to almost undetectable levels when the oviduct developed to the laying stage (over 2.88 g in weight). Similar expression patterns of qRARβ1 and β2 were also observed in the developing follicles from the prehierarchical (<2-mm diameter) to the largest preovulatory follicle. In contrast, neither qRARβ1 nor β2 mRNA exhibited developmental changes in the brain. These results suggest that RARβ may play an important role in the development of the reproductive systems of birds.

Involvement of food intake in the decreased energy retention associated with single deficiencies of lysine and Sulphur‐containing Amino acids in growing chicks

1. Growth and energy utilisation were determined in growing chicks fed ad libitum on diets deficient either in lysine (5.95 g/kg) or in sulphur-containing amino acids (SAA, 3.5 g/kg). Food intake, body weight gain, energy retained as protein and as fat, and total energy retention were significantly (P less than 0.05) reduced by single deficiencies of either lysine or SAA. 2. Another two experiments were conducted to determine if the decreased total energy retentions in chicks fed on diets deficient in lysine (experiment 3) or SAA (experiment 4) were associated with reduced food intake, by using tube-feeding to control the amount and pattern of food consumption. Chicks fed on diets deficient in lysine or SAA retained less energy as protein and more energy as fat than the control chicks. Neither total energy retention nor heat increment was affected by these deficiencies. Total energy retention was proportional to metabolisable energy (ME) intake alone. 3. It is concluded that the decreased total energy retentions caused by single deficiencies of lysine and SAA were associated with decreased food intake.

Extracellular norepinephrine in the medial hypothalamus increases during feeding in chicks: a microdialysis study.

Norepinephrinergic function in the medial hypothalamus is important for the regulation of feeding behavior in chicks as well as in rats. This study was conducted to clarify the variation of extracellular norepinephrine (NE) in the medial hypothalamus, including the paraventricular nucleus (PVN) and the ventromedial hypothalamic nucleus (VMN), during feeding behavior of layer-type chicks. To measure extracellular NE and 4-hydroxy-3-methoxyphenylglycol (MHPG), a major metabolite of NE, we used microdialysis and high-pressure liquid chromatography (HPLC) with electrochemical detection. After the collection of baseline samples, food-deprived animals were allowed access to the food for 3 h. Extracellular NE significantly increased during the first hour of access to food, and then returned to baseline levels. MHPG also increased during the feeding, but its increase continued throughout the remainder of the experiment. This study suggests that the variation of NE in the medial hypothalamus may be involved in the control of feeding in layer-type chicks.

Retinoic Acid Accelerates the Development of Reproductive Organs and Egg Production in Japanese Quail (Coturnix coturnix japonica)

Abstract The effects of retinoic acid on the development of reproductive organs and egg production in female Japanese quail (Coturnix coturnix japonica) were investigated. Female quail were fed a diet containing retinoic acid at 4 mg/kg (RA) or two diets containing retinyl acetate at 5000 IU/kg (VA1) or 14 000 IU/kg (VA2) after being fed a vitamin A-free diet for 2 wk (experiment 1). The oviduct and ovary grew more rapidly (P < 0.05) in RA-treated quail than in VA-treated quail at 5 wk of age. In addition, the body weight of RA-fed quail was also greater (P < 0.05) than that of VA-fed quail at 5 wk. The RA-treated quail laid their first eggs approximately 5 days earlier (P < 0.05) than the VA-treated quail. Furthermore, these RA-fed quail laid more eggs (P < 0.05) than those VA-fed quail during the experimental period. To confirm the results of experiment 1, a similar experiment was conducted to record the first egg and total eggs laid by quail fed VA2 or RA (experiment 2). The early onset of oviposition was again observed in the RA-treated group (P < 0.01). These results suggest that retinoic acid has a stimulating effect on the reproductive system of female Japanese quail, as has been previously shown in the reproductive system of male Japanese quail.

Regulation of Hydroxyindole-O-methyltransferase Gene Expression in Japanese Quail (Coturnix coturnix japonica)

Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.

VITAMIN A DEFICIENCY REDUCES THE RESPONSIVENESS OF PINEAL GLAND TO LIGHT IN JAPANESE QUAIL (COTURNIX JAPONICA)

Synthesis of melatonin in pineal gland is under the control of light environment. The recent finding of the presence of rhodopsin-like photopigment (pinopsin) and retinal in the avian pinealocytes has led to a hypothesis that vitamin A is involved in photoresponses of the pineal gland. We have thus analyzed the effect of vitamin A deficiency on the regulatory system of melatonin synthesis in the pineal gland of Japanese quail. Depletion of vitamin A from Japanese quails was attained by feeding them with a vitamin A-free diet supplemented with retinoic acid. In the vitamin A-deficient birds, diurnal rhythm in melatonin production persisted such that the phase of the wave was similar to that seen in the control birds. However, the amplitude of the nighttime surge of pineal melatonin was damped by vitamin A deficiency. When the control birds were briefly exposed to light at night, pineal melatonin dropped to the daytime level. In contrast, only slight decrease was observed in the vitamin A-deficient quails. The light responsiveness was restored after feeding the vitamin A-deficient quails with the control diet for 1 week. These results indicate that vitamin A plays essential roles in maintaining sufficient responsiveness of the avian pineal gland to photic input.

Effect of Arginine Deficiency on Metabolism of 14C Labeled Arginine, Glutamic Acid and Amino Acid Mixture in Growing Male Chicks

アルギニン不足飼料(適量の50%不足)を給与したヒナにおいて,飼料に由来するアミノ酸の体たんぱく質,体脂肪あるいは呼気CO2への移行がどのように変化するかを調べるために,標識アミノ酸を用いて追跡実験を実施した.8日齢白色レグホーン種雄ヒナにアルギニン適量飼料とアルギニン不足飼料を14日間等量給与した後,L-[U-14C]アルギニン塩酸塩,L-[U-14C]グルタミン酸およびL-[U-14C]アミノ酸混合物を経口投与し,体および肝臓のたんぱく質あるいは脂質,呼気CO2に移行する割合を測定した.各供試飼料を等量給与すると,アルギニン不足飼料給与により増体量と体たんぱく質蓄積量は減少し,体脂肪蓄積量は増加した.各標識アミノ酸の14Cが呼気CO2および体たんぱく質へ移行する割合は飼料のアルギニン含量によっては差がなかったが,肝臓たんぱく質への取込み割合はアルギニン不足で有意に増加した.アルギニン不足飼料の給与によって,14C-アミノ酸の体脂質への転換割合は有意に増加したが,その移行量は投与量の7%以下にしか過ぎなかった。また肝臓脂質への転換割合も増加する傾向はあったが有意な差ではなかった.これらの結果から,アルギニンの不足によって,飼料由来のアミノ酸が酸化される割合および体たんぱく質の合成に用いられる割合には変化がなく,体脂質への転換割合は促進されるものの,体脂肪蓄積量の増加に対する寄与は比較的小さいと判断した.

Carbohydrate metabolism of rumen ciliate protozoa

反芻胃内に存在するprotozoaの種構成に近い混合虫体の懸濁液を用いて,可溶性糖類の資化性について低濃度で検討を行なった結果,資化される糖はマンノースを除いて,Dasytrichaによって資化される糖に一致した.即ち,アラピノース,リボース,キシロース,メリビオースと乳糖は資化されず,ブドウ糖,果糖,マンノース,ガラクトース,セロビオース,蔗糖,麦芽糖とラフィノースは資化されて,protozoaは反芻胃内の糖類の代謝の一部に関与していることを示した.しかしマンノースについてはさらに検討する必要がある.ラフィノースの利用について,ペーパークロマトグラブィで検討して,この三糖類の一部をなしている果糖部分は資化されたが,メリビオース部分は資化されなかった.ラフィノースをこのようにして資化するprotozoaはHolotricaであると推論された.