Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.     

Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica

The vertebrate main and accessory olfactory bulbs (MOB and AOB) are the first synaptic sites in the olfactory pathways. The MOB is a cortical structure phylogenetically well conserved in its laminar structure and overall synaptic organization, while the AOB has significant species variation in size. In order to better understand signal processing in the two olfactory systems and the species differences, immunocytochemical staining and analysis were done of the neuronal expression patterns of the calcium-binding proteins calbindin D28k (CB), parvalbumin (PV), and calretinin (CR) in the MOB and AOB in a marsupial species, the gray short-tailed opossum, Monodelphis domestica. In the MOB, antibody to CB labeled periglomerular cells, superficial short axon cells / Van Gehuchten cells; antibody to PV labeled Van Gehuchten cells; and antibody to CR immunostained periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. In the AOB, CB immunoreactivity was detected in periglomerular cells and a subpopulation of granule cells; antibody to PV labeled the superficial short axon cells / Van Gehuchten cells and granule cells; and antibody to CR labeled a small number of periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. These results showed that the patterns of CB, PV, and CR expression differ in the opossum main and accessory olfactory bulbs and differ from that in other animal species. These varying patterns of neuronal immunostaining may be related to the different functions of the main and accessory olfactory bulbs and to the differing signal processing features.     

Butyrate stimulates ApoA-IV-containing lipoprotein secretion in differentiated Caco-2 cells: role in cholesterol efflux

The aim of this study was to determine: (1) whether the Short Chain Fatty Acids (SCFA) Acetate, Propionate, and Butyrate enhance the synthesis and secretion of intestinal apolipoprotein A-IV-containing lipoproteins and (2) if so, whether these particles are able to promote cholesterol efflux in vitro. For this purpose Caco-2 cells were used for their functional properties of differentiated enterocytes. They were incubated with the three SCFA (2, 4, and 8 mM) for 48 h. Only butyrate stimulated apoA-IV gene expression and this was associated with an increase in apoA-IV secretion. A nondenaturing 2D-PAGE (agarose gel was followed by PAGE) was used to identify apoA-IV-containing lipoproteins in various media, and showed that butyrate stimulated the secretion of two small HDL sized particles. The influence of these secreted particles on cholesterol efflux was investigated using incubation of media with (3)H-cholesterol-labeled Fu5AH cells. The data indicate that conditioned media from Caco-2 cells treated with butyrate resulted in an increase of 20-30% in cholesterol efflux. We conclude that butyrate may regulate apoA-IV secretion and, therefore, modulate reverse cholesterol transport.     

The obesity in bilateral ovariectomized rats is related to a decrease in the expression of leptin receptors in the brain.

We investigated the expression levels of leptin receptors in the brain of ovariectomized (OVX) rats. The mean expression level of ob mRNA in adipose tissues of OVX rats was significantly (P < 0.01) lower than that in the SHAM operation group rats, and the mean body weight of OVX rats was significantly (P < 0.01) greater than that in the SHAM group rats. However, there were no differences between serum leptin concentrations in these two groups. The mean level of leptin receptor (OB-R) mRNA expression in the brain tissue and the mean level of long form type OB-R (OB-RL) mRNA expression in the hypothalamus of the OVX rats were significantly (P < 0.05) lower than those in the SHAM group rats. These changes were cancelled by supplementation with 17 beta-estradiol in OVX rats. These results suggested that not only changes in the expression level of ob mRNA in adipose tissue and the serum leptin concentration but also changes in the OB-R mRNA in the brain are involved in the body weight increase in OVX rats and that a decrease in OB-R makes transmission of signals to suppress the amount of food intake difficult, thus leading to an increase in body weight.     

Nitrogen requirements of the didelphid marsupial Caluromys philander

The use of fruit-based diets by a small arboreal marsupial, the woolly opossum (Caluromys philander) was studied to elucidate the mechanisms used to compensate for a low dietary protein concentration. The passage of a liquid phase digesta marker (Cr-EDTA) through the gut was significantly faster when animals were fed a diet containing 0.45% N compared to that measured on a diet containing 0.90% N. The size of the gut of the two groups was similar except that the caecum of animals fed 0.45% N was significantly larger than in those animals fed 0.90% N. Animals fed a diet of 0.45% N ate significantly more food than those fed higher levels of nitrogen but there was no significant difference in the dry matter digestibility of the diet. The maintenance nitrogen requirement of the animals was 176 mg dietary N or 146 mg truly digestible nitrogen per kg metabolic body mass, with low losses of non-dietary faecal nitrogen being notable. There was no significant difference between diets in any measured parameter of urea metabolism and all animals recycled between 60% and 80% of the endogenously synthesised urea. Accepted: 22 March 2000     

Use of microsatellite polymorphisms for paternity exclusion in rhesus macaques (Macaca multatta)

A total of 224 infant rhesus macaques and their dams and potential sires in 11 multimale groups in 2 different specific pathogen free breeding colonies were screened for up to 6 different microsatellite polymorphism using cross-species PCR amplification. Observed and expected success of paternity exclusion analysis (based on gene frequencies of sires and dams in each colony) were computed by individual locus and cumulatively. Greater or less success of PEA than expected was observed at most loci due to the nonrandom distribution of genotypes between sires and dams and among breeding groups at each colony and because genotypes at different loci did not provide completely independent information about parentage. The combined success of PEA using all loci, however, was slightly greater than predicted both with and without assuming knowledge of one parent's (i.e. the dam's) genotype and was far greater than that based on protein coding loci or DNA restriction fragment length polymorphisms.     

高分辨电泳分离短串联重复序列DNA片段

采用多相缓冲系统,在成层胶T＝5%,C＝2.6%, 分离胶T＝8%,C＝5%的条件下用聚丙烯酰胺凝胶电泳对人类短串联重复序列(STR)DNA片段进行分离.其中,成层胶内主要缓冲成分为2-二羟乙基亚胺-三羟甲基甲烷(Bistris)、H₂SO₄及N、N-2(羟乙基)甘氨酸(Bicine) ; 而分离胶以Tris、H₂SO₄及2-二羟乙基亚胺-三羟甲基甲烷(Bistris)为主,构成多相缓冲系统.DNA片段在成层胶中被有效地压缩, 在分离胶内又可完全解压缩,使其按片段大小分离;从而达到提高分辨率的目的.