芦丁和IAA对绿豆黄化幼苗伸长的影响及对相关基因的RT-PCR

就芦丁(20~100μg/mL)、IAA(0.5~2.0μg/mL)及芦丁(40μg/mL)加IAA(0.5~2.0μg/mL)对绿豆黄化幼苗伸长的影响进行了研究.结果表明,芦丁对黑暗中生长的绿豆黄化幼苗下胚轴和胚根的伸长均有抑制作用,添加IAA促进了这种抑制作用.RT-PCR扩增产物的分析表明,绿豆黄化幼苗经芦丁和IAA分别处理或混合处理之后,其体内吲哚氨基酸水解酶基因、吲哚-3-甘油磷酸合成酶基因及查尔酮合成酶基因的表达在不同处理间表现出差异和多态性,芦丁处理后,其吲哚-3-甘油磷酸合成酶基因不表达.不同的RNA提取程序的比较认为,异硫氰酸胍有助于绿豆黄化幼苗RNA的提取.RT-PCR反应中不同退火温度的比较显示,较高的退火温度(60℃)有助于特异性扩增.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Experimental and clinical studies on plasma leptin in obese dogs

Leptin is a protein synthesized and secreted primarily by adipocytes, and the circulating leptin concentration is elevated in obese humans and rodents. Recently, we have established a sandwich enzyme-linked immunosorbent assay for canine leptin. In the present study, plasma leptin concentrations were measured in experimentally developed obese beagles and in clinically obese dogs. When 5 male beagles were given a high-energy diet for 3 months, all of them became obese and the plasma leptin concentration significantly increased from 2.4+/-1.2 to 4.9+/-0.9 ng/ml, positively correlating with body fat content estimated by the deuterium oxide dilution method (r=0.87). The leptin concentrations of plasma samples collected from 59 dogs in veterinary practices were compared with their body condition scores (BCS). The plasma leptin concentrations of obese dogs were 9.7+/-0.7 and 12.3+/-1.5 ng/ml at BCS=4 and BCS=5, respectively, which were significantly higher than those of optimal (BCS=3) dogs (2.7+/-0.3 ng/ml). There was no significant effect of sex and breed. A weak positive correlation (r=0.37) was found between the plasma leptin concentration and age, probably due to the lesser content of visceral fat in puppies younger than 1 year old. These results indicate that plasma leptin is a good index of adiposity in dogs regardless of breed, age and sex, and may be useful for quantitative assessment of obesity in small animal practice.     

Canine adiponectin: cDNA structure, mRNA expression in adipose tissues and reduced plasma levels in obesity

Adiponectin is a protein synthesized and secreted by adipocytes. Decreased adiponectin is responsible for insulin resistance and atherosclerosis associated with human obesity. We obtained a cDNA clone corresponding to canine adiponectin, whose nucleotide and deduced amino acid sequences were highly identical to those of other species. Adiponectin mRNA was detected in adipose tissues, but not in other tissues, of dogs. When 22 adult beagles were given a high-energy diet for 14 weeks, they became obese, showing heavier body weights, higher plasma leptin concentrations, but lower plasma adiponectin concentrations. The adiponectin concentrations of plasma samples collected from 71 dogs visiting veterinary practices were negatively correlated to plasma leptin concentrations, being lower in obese than non-obese dogs. These results are compatible with those reported in other species, and suggest that adiponectin is an index of adiposity and a target molecule for studies on diseases associated with obesity in dogs.     

Antidiabetic activities of chalcones isolated from a Japanese Herb, Angelica keiskei

Diabetes mellitus is a chronic disease that is characterized by hyperglycemia caused by insufficient insulin action. We have explored the edible ingredients from folk medicines in Japan that contain substances complementing insulin action, such as the induction of adipocyte differentiation and the enhancement of glucose uptake. We eventually found that the ethanol extract from a Japanese herb "Ashitaba", Angelica keiskei, contained two major chalcones of 4-hydroxyderricin (4-HD) and xanthoangelol that showed strong insulin-like activities via a pathway independent of the peroxisome proliferator-activated receptor-gamma activation. The 4-HD especially showed the preventive effects on the progression of diabetes in genetically diabetic KK-Ay mice.     

Establishment of an Invasive Prostate Cancer Model in Transgenic Rats by Intermittent Testosterone Administration

We have established a transgenic rat for adenocarcinoma of the prostate (TRAP) model that features uniform adenocarcinoma development in prostatic lobes at high incidence within a short experimental period. However, no invasive carcinomas with reactive stroma characteristics similar to those in man were observed. We therefore have focused on a new model for invasive carcinoma of the prostate using TRAP rats. In experiment 1, male TRAP rats in groups 1 and 2 were treated with orchiectomy at day 0 of the experiment. Rats in groups 1–3 underwent testosterone propionate (TP) implantation from weeks 1 to 4 and from weeks 6 to 16. Rats in groups 1 and 3 were given 3,2’-dimethyl-4-aminobiphenyl (DMAB) after TP implantation. The rats of group 4 served as controls. In experiment 2, the rats were divided into three groups, none of which received DMAB or orchiectomy, treated with TP continuously or with the treatment withdrawn once or twice. In experiment 1, invasive adenocarcinomas with abundant collagenous stroma were found in the dorsolateral and anterior prostate, some of which showed perineural space invasion at week 16. The number of invasive carcinoma foci was most frequent in group 3. In experiment 2, invasive adenocarcinoma development in the lateral prostates was correlated with the number of TP administration/withdrawal cycles. In conclusion, our newly established rat model for invasive adenocarcinoma of the prostate could serve as a useful preclinical model for evaluating the in vivo efficacy of preventive and therapeutic agents targeting of the tumor microenvironment.     

Correlation between plasma leptin concentration and body fat content in dogs

OBJECTIVE: To evaluate the relationship between plasma leptin concentration and body fat content in dogs. ANIMALS: 20 spayed female Beagles that were 10 months old at the start of the experiment. PROCEDURE: Dogs were kept under regulated feeding and exercise conditions for 21 weeks, resulting in a wide range of body weights, body condition scores (BCS), and subcutaneous thicknesses. Plasma leptin concentration was measured by use of a canine leptin-specific ELISA test to evaluate its correlation to body fat content estimated by the deuterium oxide dilution method. Plasma concentrations of glucose, cholesterol, triglycerides (TG), and nonesterified fatty acids (NEFA) were also measured. RESULTS: Body fat content (9 to 60% of body weight) was positively and closely correlated (r = 0.920; n = 20; P < 0.001) to plasma leptin concentration (0.67 to 8.06 ng/ml), compared with other variables (ie, glucose, cholesterol, TG, and NEFA; r = 0.142, 0.412, 0.074, and 0.182, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The positive relationship between plasma leptin concentration and body fat content in dogs was similar to correlations reported for humans and rodents, suggesting that plasma leptin is a quantitative marker of adiposity in dogs.     

COMPUTED TOMOGRAPHIC ASSESSMENT OF BODY FAT IN BEAGLES

Obesity is the most common nutritional disorder in small animals. To establish a computed tomographic (CT) method for assessment of visceral and subcutaneous fat content in the dog, CT analysis was performed in normal and obese beagles. Fat area was measured by the level detection method at varied attenuation ranges and compared with body fat content estimated by the deuterium oxide dilution method. Fat area measured at L3 using the attenuation range of -135/-105 Hounsfield unit had the best correlation with body fat content (r = 0.98). Regional fat distribution was almost the same between normal and obese dogs, with more fat accumulation at L1-S1 than T10-T13. Moreover, visceral and subcutaneous fat area could be estimated separately. This CT method may contribute to both the clinical diagnosis and the study of canine obesity, especially for studies in the relationship between body fat distribution and obesity-associated diseases.