Quantitative autoradiographic assessment of 55Fe-RBC distribution in rat brain

A simple in vivo technique of labeling erythrocytes (RBCs) with 55Fe was developed for quantitative autoradiography (QAR). This procedure involved injecting 5-6 ml of [55Fe]ferrous citrate solution (1 mCi/ml) intraperitoneally into donor rats. The number of labeled RBCs reached a maximum at around 7 days and declined very slowly thereafter. Labeled RBCs were harvested from donor rats and used for RBC volume measurement in awake rats. Brain radioactivity was assayed by QAR, which yielded spatial resolution of greater than 50 microns. Tight nearly irreversible binding of 55Fe to RBCs was found in vivo and in vitro. More than 99.5% of the 55Fe in the blood of donor rats was bound to RBCs. Because of this, labeled blood can be taken from donors and injected into recipients without further preparation. The tissue absorption of 55Fe emissions was the same in gray and white matter. Microvascular RBC volumes measured with 55Fe-labeled RBCs agreed with those assayed with 51Cr-labeled RBCs for many, but not all, brain areas. In conclusion, 55Fe-RBCs can be readily prepared by this technique and accurately quantitated in brain tissue by QAR.     

Enzyme distribution and transport activities in electrophoretically isolated fractions of the muscle sarcotubular system

A simple electrophoretic method is introduced allowing to isolate five fractions of skeletal muscle ST-system vesicles. In a previous study differences in lipid content, 3H-ouabain binding and in presence of triads in individual fractions (Lehotsky et. al. 1986) were analysed. In the present study biochemical characterization was extended, and (in accordance with previous results) major differences were observed to exist between fraction 1 and fractions 3 and 4. SDS-PAGE showed that fractions 3 and 4 were enriched in a protein with m.w. 100 kD, these fractions showing the highest specific activities of (Mg2+ + Ca2+)-ATPase and oxalate-supported Ca2+-uptake; activities of Mg2+-ATPase and surface membrane marker enzymes were the lowest in these fractions. On the other hand, in fraction 1 the highest activities of Mg2+-ATPase and marker enzymes of the surface membrane were observed together with a decreased content of the 100 kD protein and activities of Ca2+ transport. It could be concluded that the method is suitable to differentiate between relatively pure SR (fractions 3 and 4) and fractions rich in sarcolemma or T-tubules components (fractions 1 and 5).     

Electron microscopic and polarographic study of rat liver mitochondria during freezing and thawing in sucrose and saline media

Results are presented of studying structural and functional changes in rat liver mitochondria during freezing-thawing in sucrose and saline medium. It is found that maximal disturbances in the structural and functional state of mitochondria are observed in the saline medium.     

Quantitative characteristics of glutamate transport in rat liver mitochondria

1. The kinetics of glutamate transport into mitochondria were determined by using Bromocresol Purple to terminate the transport process. 2. Glutamate transport was found to have a V(max.) of 9.1nmol/min per mg of protein at pH6.9 and 20 degrees C; the K(m) for glutamate was 4mm. 3. The rate of glutamate deamination in intact mitochondria was tenfold slower than in disrupted mitochondria. 4. These results suggest that glutamate deamination may be controlled by the rate of glutamate transport. Possible consequences of these findings are discussed.     

大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法     

Quantitative characteristics of oxygen tension distribution in arterioles,capillaries, and venules of the rat brain cortex in normoxemia

Distribution of the oxygen tension in the rat brain venules in normoxemia was characteristic by its obvious heterogeneity. The data obtained suggest a tissue diffuse shunting of oxygen in the brain cortex of rats in normoxenia.     

Cholinesterase activity of capillaries in the rat brain. A light and electron microscopic study

Synopsis The presence of cholinesterase activity in association with capillaries of the central nervous system was investigated in the rat by means of both light and electron microscopic methods. Throughout most of the rat brain, the smaller blood vessels stain intensely for butyrylcholinesterase activity. In some areas, such as the commissural nucleus of the vagus and parts of the medial thalamus, the capillaries possess both acetylcholinesterase and butyrylcholinesterase activity. Blood vessels in those structures which lie outside the blood-brain barrier are completely devoid of cholinesterase activity. The electron microscope reveals that reaction product occurs within the matrix of the basement membrane, in the intermembranous space of the endothelial nuclear envelope and occasionally in the endothelial granular endoplasmic reticulum. It is suggested that the presence of cholinesterase within the basement membrane of brain capillaries is evidence of the role that the basement membrane may play in transfer mechanisms.     

Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain

1. Subcellular fractions and myelin were isolated from developing and adult rat brain. 2. Measurements of chemical composition and enzyme activities indicate the presence of a second myelin-like fraction mainly in the brain of developing rats. 3. This membrane fraction has a different lipid composition from myelin, but resembles myelin in its content of phosphohydrolase and aminopeptidase activity. 4. It is suggested that the second myelin-like fraction may be a submicrosomal contaminant or it may be derived from oligodendroglial plasma membrane during myelinogenesis.     

Correlative scanning-transmission electron microscopic examination of the perinatal rat brain

Summary The ultrastructural organization of the perinatal hypothalamus and the dynamics of neuronal and ependymal growth and plasticity were examined in this investigation. The brains of fetal rats 16, 17 and 18 days in utero and those of postnatal rats 1–16 days post partum were fixed with aldehyde fixatives and prepared for combined SEM/TEM analysis. By day 17 in utero the ventricular (ependymal) surfaces of the fetal thalamic wall, cerebral vesicle and rhomboid fossa were relatively well differentiated with cilia and microvilli. Type II histiocytes were the first supraependymal cell to appear upon the ventricular lumen and were evident by day 17 in utero. In contrast, the apical surfaces of tanycytes of the infundibular recess as well as those of most other circumventricular organs were poorly differentiated and unremarkable. Tanycytes of the infundibular recess exhibited a simple hexagonal mosaic pattern of apposed plasmalemmata and even by day 1 post partum few cilia or microvilli were evident.By day 5–6 post partum Type I supraependymal neurons and axonal processes began to make their appearance with some emerging from the underlying parenchyma of the median eminence. By day 16 post partum the ventricular surface of the infundibular recess was comparable with that of the adult.The Type I supraependymal neurons are remarkably similar in their ultrastructural organization with parvicellular neurosecretory neurons elsewhere in the endocrine hypothalamus. Their emergence at day 5–6 post partum suggests a possible correlation with the critical period of sexual differentiation and a potential receptor role for this cell line. On the contrary this phenomenon may simply be a developmental anomaly. Nonetheless, the mergence of such elements upon the lumen of the third cerebral ventricle underscores a remarkable degree of neuronal plasticity in the perinatal hypothalamus.Supported by USPHS Program Project Grant NS 11642-04 and USPHS-BRSG Grant RR-05403.The authors wish to thank N. Kutryeff for her excellent technical assistance