新井 敏郎

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuronspecific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of pre'dnisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis. (c) 2006 Elsevier B.V. All rights reserved.

To investigate the effect of Agaricus mushroom meal on the energy metabolism in animal tissues; plasma glucose, triglyceride, cholesterol and immunoreactive insulin (IRI) concentrations and activities of enzymes related to energy metabolism in plasma and peripheral leukocytes were measured in Japanese Black Wagyu x Holstein 171 calves supplemented with Agaricus blazei Murill (A. blazei) extract in milk-replacer at the dose of 60 g/head/day for 4 weeks. Activities of malate dehydrogenase and aspartate aminotransferase in cytosol and glutamate dehydrogenase in mitochondria, and the malate dehydrogenase/lactate dehydrogenase ratio in cytosol in peripheral leukocytes of calves with A. blazei were significantly higher than those in control calves without A. blazei. It was concluded that supplementation of Agaricus mushroom meal extract was effective in activation of enzymes related to energy metabolism in peripheral leukocytes of calves. (c) 2006 Elsevier Ltd. All rights reserved.

日本獣医生命科学大学の「ゲノム・プロテオーム解析による予防獣医学の展開」プロジェクトが平成17年度文部科学省学術研究高度化推進事業・学術フロンティア推進事業の拠点の一つに選定された。このプロジェクトは獣医学,畜産学,栄養学,免疫学,疾病予防学を包含する研究で,ゲノム,プロテオーム解析により動物の疾病の新たな診断・予防システムの確立を目指すものである。疾病の原因となる因子の検索や発症メカニズムのゲノム,プロテオームおよびメタボローム解析技術を駆使した微量・迅速定量技術の確立により,疾病の早期診断を基にした疾病の予防や軽症化を図る「予防獣医学」を展開していく予定である。この研究の過程で生み出される成果は医学,畜産学領域をはじめとして多くの学問領域に有用な知見を提供し,生命科学研究の進展に寄与するものと確信している。

Plasma metabolite and immunoreactive insulin concentrations and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in growing Holstein calves. A ratio of girth of abdomen divided by girth of thorax (A/T ratio) of calves was significantly elevated after weaning, and the A/T ratio maybe a good indicator to evaluate rumen development. Plasma glucose and free fatty acid concentrations were changed in calves accompanying change in feeding. Activities of lactate dehydrogenase with pyruvate as substrate (LDH-P) and hexokinase (HK) in cytosolic fractions of peripheral leukocytes decreased significantly after weaning the calves reflecting the change of energy source from milk replacer with high percentages of fat and glucose and lactose as absorbable carbohydrate to pelleted feed containing starch as less absorbable carbohydrate and roughage. Some peripheral leukocyte enzymes such as LDH and HK may be good indicators to evaluate changes in energy metabolism of growing calves. (c) 2005 Elsevier Ltd. All rights reserved.

We have established an inbred line of mice deficient in insulin receptor substrate 2 (IRS2) on a C57BL/6J Jcl genetic background (B6J-IRS2(-1-) mice) as an animal model for typical type 2 diabetes mellitus (DM). We investigated the effect of age and sex on glucose tolerance and insulin resistance and on the activities of enzymes related to lipid metabolism in the liver and skeletal muscle of B6J-IRS2(-/-) mice. Glucose tolerance tests (GTT), insulin tolerance tests (ITT), and sampling for chemical analysis were performed at ages of 6, 14, and 24 wk. GTT showed that both genders of B6J-IRs2(-/-) mice had impaired glucose tolerance at the ages of 6 and 14 wk, whereas 24-wk-old female B6J-IRs2(-/-) mice showed glucose tolerance almost comparable to that of wild-type mice; 24-wk-old male B6J-IRs2(-/-) mice still showed impaired glucose tolerance. ITT revealed that both male and female B6J-IRS2(-/-) mice remained insulin-resistant at all time points. Hepatic lipogenetic enzyme activities were higher in B6J-IRS2(-/-) mice than in wild-type mice at 6, 14 and 24 wk of age. In addition, plasma glucose, triglycericle, free fatty acid, total cholesterol, and insulin concentrations in B6J-IRS2(-/-) mice were significantly higher than those in wild-type mice at most time points; plasma triglycerides in 14-wk-old B6J-IRS2(-/-) mice were lower than those of wild-type mice. These findings suggest that young B6J-IRS2(-/-) mice are useful as type 2 DM models.

Plasma glucose and immunoreactive insulin (IRI) concentrations and activities of enzymes related to glucose metabolism in livers were measured in dogs and cats. Nucleotide sequences of the conserved region of glucokinase (GK) cDNA that contained ATP- and glucose-binding domains were determined in canine liver and feline pancreas for design of the species-specific oligonucleotide primers for reverse transcription-polymerase chain reaction (RT-PCR) analysis. There were no significant differences in plasma glucose and IRI concentrations between dogs and cats. In feline liver, although GK activities were not detected, activities of hexokinase, fructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly higher than those in canine liver. The partial sequences of canine liver GK and feline pancreas GK cDNA were respectively 88% and 89% identical with the rat liver GK cDNA. Expression of GK gene was observed in canine liver and pancreas and feline pancreas with RT-PCR using species specific primers based on the cDNA sequences.

Alterations in the activities of enzymes related to energy metabolism in canine lymphoma cells were investigated. Cytosolic pyruvate kinase (PK) and mitochondrial malate dehydrogenase (MDH) activities in lymphoma cells were significantly higher than those in lymphocytes obtained from lymph nodes of healthy dogs, whereas cytosolic lactate dehydrogenase (LDH) activity was significantly lower in lymphoma cells. The cytosolic M/L ratio (MDH activity/LDH activity), which is considered to be a good indicator of energy metabolism related to glucose utilization in animal tissues, was significantly higher in lymphoma cells than in the normal lymphocytes.

The activities of the enzymes in the malate-aspartate shuttle were measured in peripheral leucocytes of spontaneous type 1 diabetic dogs and cats treated with insulin injections. In the diabetic does and cats, fasting plasma glucose concentrations were three or fourfold greater than the control levels in spite of insulin injections and the activities of cytosolic malate dehydrogenase (MDH), one of pivotal enzymes in the malate-aspartate shuttle, were remarkably lower than the controls. Depressed expression of cytosolic MDH mRNA was confirmed by RT-PCR analysis in the diabetic animals. The cytosolic ratio of MDH/lactate dehydrogenase (LDH) activity (M/L ratio) in leucocytes of the diabetic animals was significantly lower than that of normal control animals. The smaller M/L ratio appeared to reflect depression of energy metabolism in the diabetic animals. Intrinsically lower and further decreased MDH activities may be factors that induce insulin resistance observed in diabetic cats. (C) 2004 Elsevier Ltd. All rights reserved.

Plasma metabolites and immunoreactive insulin (IRI) concentrations and enzyme activities of some types of peripheral leucocytes were measured to clarify one aspect of the differences in nutrient metabolism between dogs and cats. There were no significant differences in plasma concentrations of glucose.. triglyceride, free fatty acids and IRI between dogs and cats. Higher total cholesterol and lower HDL cholesterol concentrations were observed in feline plasma, and H/T ratio (HDL/total cholesterol concentrations) was significantly lower than that in canine plasma. The cytosolic activities of fructokinase (FK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) were significantly higher and the activities of cytosolic malate dehydrogenase (MDH) and mitochondrial glutamate dehydrogenase (GLDH) were significantly lower in feline than those in canine leucocytes. Higher activities of FK, PK and G6PD, which regulate the rate of biosynthesis of fatty acids, may reflect the different characteristics in nutrient metabolism in feline tissues from canine tissues. (C) 2004 Elsevier Ltd. All rights reserved.

Transgenic mice hemizygously carrying human c-Ha-ras proto-oncogene, Tg-rasH2 show Very sensitive and facilitated carcinogenicity to various carcinogens. In this study, activities of certain enzymes related to drug metabolism and energy metabolism were measured in microsome and cytosol fractions of livers of Tg-rasH2 mice and their wild type littermates with both sexes treated with 3-methylcholanthrene (MC) and phenobarbital (PB). Aminopyrine N-demethylase activities increased significantly in livers of all mice treated with PB. MC and PB treatments induced significant increases in activities of UDP-glucuronosyltransferase and S-adenosyl homocysteinase compared to those in the non-treated groups in microsome fractions from all mice. In cytosol fractions of livers of all mice, glutathione S-transferase activity was significantly induced in the PB treated groups. There were no significant differences in activities of lactate dehydrogenase, glucose g-phosphate dehydrogenase, pyruvate kinase and glucose 6-phosphatase related to energy metabolism in livers and kidneys among all mice. Tg-rasH2 mice showed stable activities of enzymes related to drug detoxication and energy metabolism similar to those of non-transgenic mice. These results suggest that the human c-Ha-ras transgene may not affect drug metabolism-related enzymes, and the facilitated carcinogenic response in the Tg-rasH2 mouse is not due to these enzymatic disorders.

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 mu g/ml chondroitin sulfate A (CS), 5 mu g/ml hyaluronic acid (HA), or 5 mu g/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.

Activities of enzymes related to glucose metabolism were measured in canine and feline liver. There were no significant differences in plasma glucose and immunoreactive insulin concentrations between dogs and cats. Glucokinase activities were absent in feline liver, however, activities of other glycolytic enzymes such as hexokinase, phosphofructokinase and pyruvate kinase, were significantly higher than those in canine livers. Activities of rate limiting enzymes of gluconeogenesis such as pyruvate carboxylase, fructose-1,6-bisphosphatase and glucose-6-phosphatase in feline livers were significantly higher than those in canine livers. (C) 1999 Harcourt Publishers Limited.

Ingestive behavior was activated in male rats by intraoral intake and intake from a bottle of l-M solution of sucrose. Intraperitoneal injection of p-chloroamphetamine (PCA), releasing central 5-hydroxytryptamine (5-HT) from serotonergic nerve terminals, inhibited ingestion of the sucrose solution. Significant inhibition of sucrose intake by PCA was observed at 1.25 and 2.5 mg/kg dose in a bottle intake test, and at 5.0 mg/kg dose in an intraoral intake test. These findings suggested that 1.25 and 5.0 mg/kg of PCA suppressed appetitive ingestive behavior and consummatory ingestive behavior in male rats, respectively.

Hyaluronidase, acrosin and N-acetylhexosaminidase activities were examined in sperm collected from 12 beagle dogs and in culture medium after 0.5 hr and 7 hr of sperm incubation. The activities of the three enzymes were significantly higher at 7 hr than at 0.5 hr (P<0.05, 0.01), and the increases were associated with sperm capacitation. It was considered that the three enzymes in the dog sperm are related to fertilization by reason of the findings of the release of these enzymes from the sperm into the medium after 7 hr of incubation.

草食性ハタネズミとC57BL/6Jマウスの赤血球および諸組織におけるグルタチオンペルオキシダーゼ (GSH-Px) 活性を比較検討した。ハタネズミ, マウスともに, 肝臓, 腎臓, 赤血球のGSH-Px活性が, 脂肪組織, 膵臓, 骨格筋に比較して高かった。また, ハタネズミのGSH-Px活性は, 肝臓, 腎臓, 赤血球および脳で, マウスに比べ低い値を示した。

Activities of hepatic ATP-citrate lyase and acetyl CoA carboxylase, lipogenetic key enzymes, were measured in herbivorous voles and C 57 BL/6 J mice. Hepatic ATP citrate lyase and acetyl CoA carboxylase activities in voles were very low, under one fifth and one half of those in C 57 BL/6 J mice, respectively. It is considered to be one of characteristics as a herbivore that hepatic lipogenetic ability in voles is considerably low as compared to that in mice.

草食性ハタネズミとC57BL/6Jマウスにおいて, 脂肪酸合成の律速酵素である肝ATP-クエン酸リアーゼとアセチルCoAカルボキシラーゼの両酵素活性を測定し, 比較検討を行った。ハタネズミの肝ATP-クエン酸リアーゼ, アセチルCoAカルボキシラーゼ活性もマウスのそれに比べ各々1/5, 1/2以下と著しく低かった。ハタネズミの脂肪酸合成能力はマウスに比べ著しく低く, これは, 草食動物の特性のひとつと考えられた。

やせ型糖尿病 (NOD) マウス血清中の抗核抗体に対する核蛋白質抗原について調べた。C57BL/6JおよびNODマウスの肝凍結切片を抗原として用いた間接蛍光抗体法により, 尿糖陽性の雌NODマウス (20週齢) 血清中に抗核抗体が検出された。この血清を用いて, C57BL/6Jマウスの肝から分離一抽出した可溶化核蛋白質抗原をSDS-PAGE Western immunoblotting法により検索した結果, 分子量65000, 32000, 26000の蛋白質がNODマウス血清中の抗核抗体と特異的に反応した。

MSA投与により作出した肥満性糖尿病KK雄マウスを, 若齢群 (12週齢) と高齢群 (35週齢) に分け, 去勢およびアンドロゲン投与を行い, 糖尿病発症におよぼすアンドロゲンの影響を調べた。血中アンドロゲンレベルは, マウス涙液中の雄性ホルモン依存性蛋白質 (Mtp-M) の消長を, ゲル電気泳動法で観察することにより推定した。若齢マウスでは去勢後一時的に尿糖が消失したが, 再び尿糖が出現した。Mtp-Mは去勢により弱化が認められたが, 消失はしなかった。これに対し, 高齢マウスでは去勢後に尿糖が陰転し, 血糖値は去勢前と比べ著しく低下した。また, Mtp-Mは完全に消失した。いっぽう, 去勢後, 尿糖が陰転した高齢マウスにdehydroepiandrosterone (DHEA) を投与した結果, 再び尿糖の陽転と血糖値の上昇 (98.8mg/dl→198.3mg/dl) がみられ, これに伴ってMtp-Mの出現が認められた。以上の結果から, MSA処理KKマウスの糖尿病発症には, アンドロゲンが重要な役割を果たしていることが強く示唆された。