Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA.

Expression of cell-cycle-regulating genes in the development of atherosclerosis in Japanese quail (Coturnix japonica).

The levels of mRNA expression in regulatory genes that are involved in the pathological changes of aortic atherosclerotic and fibroblastic intimal thickening was investigated in Japanese quail. The quail were divided into a control diet group and an atherogenic diet group. The quail were euthanized at 2, 4, 8, and 12 wk after consuming either a control diet or an atherogenic diet. Thereafter, both histological and immunohistochemical studies and mRNA expression analysis of the cell-cycle-regulating genes in aortic atherosclerotic lesions were performed on selected ascending aortas and their large branches. In the atherogenic diet group, aortic lipid-containing intimal and atheromatous lesions were seen mainly at 8 and 12 wk, respectively. Semiquantitative reverse-transcription PCR was used to analyze the alterations of mRNA expression on the development of atherosclerotic lesions. Messenger RNA expression of the c-fos and c-src genes showed peak levels at 8 wk in the atherogenic diet group. However, no significant alteration of c-jun mRNA expression was noted during the entire experimental period. According to the progression of aortic atherosclerotic lesions, c-myc mRNA expression in the atherogenic diet group increased chronologically, and the highest level was observed at 12 wk. Alterations in mRNA expression of proliferating cell nuclear antigen and the p27 gene were similar to that of c-myc. The levels of c-myc, proliferating cell nuclear antigen, and p27 mRNA expression was significantly correlated with the degree of aortic atherosclerotic lesion development at 12 wk in our experiment.

Prenatal and postnatal maternal effects on body weight in cross-fostering experiment on two subspecies of mice.

A cross-fostering experiment was conducted on two quite distinct subspecies of mice, domesticated laboratory mouse of CF#1 (Mus musculus domesticus) and Yonakuni wild mouse (Yk, Mus musculus molossinus yonakuni), to estimate the prenatal and postnatal maternal effects on body weight of offspring. Mating was done between subspecies, two or three females being mated to a male at nine-ten weeks of age. Two dams of different subspecies that littered at the same day were used as a group of foster dams. Litters were standardized to six young mice in order that a dam nursed three mice of her own litter and three mice from that of another subspecies dam. The litters were weaned at 3 weeks of age. The body weight of individual mice was determined at 1, 3, 6 and 10 weeks of age. The result demonstrated that prenatal maternal effects were more important than postnatal maternal effects in contributing to the variation in body weight at all ages examined. Prenatal maternal effects accounted for 61-96% and 35-92% of total variance in males and females, respectively; whereas postnatal effects accounted for 1-7% for males and 3-23% for females. Analysis for between postnatal within prenatal, and between prenatal within postnatal indicated that expression of the body weight of offspring was limited by the genetic type of their prenatal dam and influenced by the postnatal environment of nursing dam. The greatest body weight was attained by offspring born to prenatal CF#1 dams and nursed by postnatal CF#1 dams, followed by CF#1 offspring born to CF#1 dams and nursed by Yk dams, Yk offspring born to Yk dams and nursed by CF#1 dams and the lightest ones were Yk offspring born to Yk dams and nursed by Yk dams.

Diallel cross analysis of body weight in subspecies of mice.

A complete 4 x 4 diallel cross of CF#1 (C), C57BL/6NCrj (B) C3H/HeNCrj (H) and Yonakuni wild mice (Y, Mus musculus molossinus yonakuni) has been conducted to estimate the effects of sex, degree of heterosis, general combining ability (gca), specific combining ability (sca), maternal ability, and reciprocal cross on body weight at 1 (Wk1), 3 (Wk3), 6 (Wk6) and 10 (Wk10) weeks of age. A least squares analysis was performed on 828 mice and all sources of variation showed significant effects (P < 0.01) on body weight but not sex at Wk1 (P > 0.05). Males were heavier than females (P < 0.01) at Wk3, Wk6 and Wk10. C and Y were the heaviest and lightest in body weight, whereas H and B were intermediate. Differences in body weight were observed between linebred and linecross at all ages studied: 6.57%, 10.22%, 8.70% and 5.89% heterosis for the respective ages. The degree of gca and maternal effects can be ranked as C > H > B > Y. Crossing between C and H had greater sca than other combinations at all ages studied, whereas B x Y had the smallest. Mean body weight of the offspring from two-line reciprocal cross differed according to their dam. A relatively large proportion of additive genetic effects in contributing to the variation in offspring body weight was indicated.

Changes in single unit activity in the lateral hypothalamic area of goats during feeding.

In order to clarify the location of feeding centers in the ruminant brain, this study used a single-unit activity (SUA) recording electrode to investigate the existence of appetite-regulating neurons in the lateral hypothalamic area (LHA) in goats. Seven male Japanese Saanen goats were used in the experiment. The animals were fed twice daily, once in the morning (1000 to 1200) with 1.5 kg of roughly crushed alfalfa hay cubes, and once in the afternoon (1600 to 1800) with 200 g of commercial ground concentrate feed. The animals were allowed free access to drinking water. In this study, the animals were surgically operated on to position the recording electrode in the LHA. Recordings of SUA were carried out continuously over a 2.25-h period beginning 15 min prior to the commencement of morning feeding. The eating rates of crushed alfalfa hay cubes were highest 10 min after feeding commencement, but decreased sharply by the time 40 min had elapsed. The cumulative feed intake after the completion of the 2-h feeding period was 1164 +/- 38 g. The cumulative water intake upon the conclusion of the 2-h feeding period was 2422 +/- 107 mL. This study recorded 31 units, of which five showed a response to feeding and altered their firing rates. In response to a sharp increase in eating rates, all five units increased their firing rates to a level higher than that of prefeeding (P < 0.05). As the animals reached a level of satiety (eating rates declined to very low levels), firing of units I and II stopped completely, while the firing rates of units III, IV, and V decreased. Examination of a serial histological section confirmed that the five units in which changes in firing rates with feeding were observed were all located in the dorsolateral hypothalamic area close to the fornix. The LHA neurons recorded in this experiment characteristically showed neuronal activity increases at high levels of feeding, but decreases at low levels. The results suggest that there are cells located in the LHA of goats that are active in the physiological regulation of hay (dry forage) intake.

PP077. Effect of maternal antioxidant concentrations on expressions of angiogenesis-related genes in the villous trophoblasts from early pregnancy.

INTRODUCTION: The pathogenesis of preeclampsia is associated with oxidative stress of villous trophoblasts in early pregnancy. We have reported that antioxidant supplementations from 12weeks of gestation could prevent mild preeclampsia in pregnant women with oxidative stress. OBJECTIVES: To prove the effect of maternal antioxidant concentrations on expressions of angiogenesis-related genes in the villous trophoblasts from early pregnancy. METHODS: Villous tissue and blood samples were obtained from pregnant women who had artificial abortions between 6 and 11weeks of gestation. RNA was extracted from villous tissue, and gene expression levels of fms-like tyrosine kinase-1 (FLT-1), endoglin (ENG), and placental growth factor (PGF) in villous tissue were measured by reverse transcription polymerase chain reaction. Serum vitamin C and E concentrations were measured by enzyme-linked immunosorbent assay. This study was approved by the Ethics Committee of Showa University Hospital. RESULTS: Negative correlations between maternal serum vitamin C levels and gene expressions of ENG (r=-0.856, p=0.007) and FLT-1 (r=-0.898, p=0.002) in the villous trophoblasts were observed. There was no correlation of maternal serum vitamin E concentration with any other gene expression in the villous trophoblasts. CONCLUSION: It is suggested that maternal oxidative stress may increase the gene expressions of anti-angiogenic factors in villous trophoblasts of early gestation, which may lead to the pathogenesis of preeclampsia.