CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between-age differences in the activity of cyclic AMP-dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent K_a, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP-dependent phosphorylation system in the rat brain does not change during advanced aging.     

SUBCELLULAR DISTRIBUTION OF A HEAT-STABLE PROTEIN INHIBITOR OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN RAT BRAIN

Abstract— Cyclic AMP (cAMP)-dependent protein kinase catalyzes the phosphorylation of polypeptidic serine and threonine residues according to the following chemical equation: ATP + protein → phospho-protein + ADP. A heat stable, trypsin labile factor present in brain, skeletal muscle and other tissues inhibits enzymatic phosphorylation of some proteins and enhances that of others. Since brain is one of the richest sources of adenylate cyclase, cAMP, cAMP-dependent protein kinase and the heat stable protein kinase inhibitor and because they may play a role in neurotransmission, an investigation of the subcellular distribution of the heat stable factor in rat brain was undertaken. Although present in the nuclear, mitochondrial and microsomal fractions, the highest activity of protein kinase inhibitor is in the soluble fraction: its activity parallels that of the cytoplasmic enzyme marker, lactate dehydro-genase. The inhibitory activity is also found in the synaptosome or pinched-off nerve ending fraction. Following osmotic lysis of this fraction, about 90% of the factor occurs in the soluble fraction. On the other hand, only 40% of the cAMP-dependent protein kinase is solubilized and 60% remains membrane-bound. Using this membrane-bound protein kinase, phosphorylation of endogenous substrate is unaltered by inhibitor, but phosphorylation of added histone substrate is decreased.     

CYCLIC NUCLEOTIDES and PROTEIN KINASE ACTIVITY IN THE RAT BRAIN POSTMORTEM

Abstract— The components of the cyclic nucleotide system were studied in rat brain at various times after death to determine the stability of the system postmortem. The concentration of cyclic AMP in 4 brain regions was highest 10 min after death and declined to stable levels within 6 h after death. At this time concentrations approximated those normally seen in vitro. The concentration of cyclic GMP in brain regions fell markedly postmortem and was undetectable 6 or 16 h after death. Nor-epinephrine-stimulated cyclic AMP accumulation measured in vitro in slices of the cerebral cortex was the same 10 min and 16 h postmortem. In the cerebellum, however, accumulated levels of cyclic AMP were lower 16 h postmortem, although the degree of stimulation elicited by norepinephrine was the same 10 min and 16 h after death. Cyclic AMP-dependent and independent protein kinase activities were detected in the rat brain at all times after death. There was no change in the cyclic AMP-dependent enzyme activity postmortem, but activity in the absence of cyclic AMP was significantly higher immediately after death compared to 6 or 16 h postmortem. These studies indicate that it should be possible to study the cyclic AMP system in human brain tissue obtained at autopsy.     

RNA POLYMERASE ACTIVITY IN VARIOUS CLASSES OF NUCLEI FROM DIFFERENT REGIONS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

—The changes in the wet weight and the numbers of cell nuclei recovered from the cerebral hemispheres, the cerebellum and the brain stem of rats from the period of 5–30 days after birth have been determined. In parallel a study has been made of the RNA polymerase activity, both in the unfractionated nuclei from these regions and in the nuclei separated by zonal centrifugation. In general there is a considerable decline in activity during this period, which occurs in all class of nuclei although not to the same extent. The most dense nuclei from the cerebellum retain relatively high activity at 20 days after birth, possibly due to the contribution of the microneuronal nuclei.     

IDENTIFICATION OF A CYCLIC AMP-DEPENDENT PROTEIN KINASE IN THE DINOFLAGELLATE AMPHIDINIUM OPERCULATUM

Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle.     

NUCLEAR RIBONUCLEASE ACTIVITIES OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— The activities of alkaline and acidic RNAses were determined in soluble and insoluble fractions from nuclei of brain hemispheres of rats, aged from 1 day to adult. The activities increased rapidly and reached a maximum, at 30 days, of about 10 times (alkaline RNAsel or 5 times (acidic RNAse) that seen at day 1.     

促甲状腺激素释放激素受体-1(TRH-R1)蛋白在大鼠出生后睾丸不同发育阶段的表达和定位

目的研究促甲状腺激素释放激素受体-1(thyrotrophin-releasing hormone receptor type-1, TRH-R1)在大鼠睾丸出生后不同发育阶段的表达,探讨其在生殖发育调节中的作用.方法应用蛋白质免疫印迹杂交技术以及免疫组织化学ABC法检测TRH-R1在8d、15d、20d、35d、60d和90d大鼠睾丸中的表达和定位,并结合图像分析技术对免疫组化结果进行统计学分析观察其在发育过程中的变化.结果免疫印迹杂交发现TRH-R1蛋白表达于15d以后各阶段的大鼠睾丸;而运用免疫组化在第8d即检测到TRH-R1的表达,以后发育过程中的各个阶段均有阳性反应细胞, TRH-R1定位于大鼠睾丸的间质细胞;免疫反应阳性物均位于胞膜和胞质,胞核区为阴性;图像分析结果表明,随着大鼠睾丸的发育,TRH-R1表达量呈增多趋势,且具有统计学差异(P＜0.01).结论本实验证明TRH-R1在出生后8d大鼠的睾丸内即有表达,并持续表达于其后各个发育阶段;TRH-R1定位于睾丸的间质细胞,其表达量随着增龄变化呈增多趋势,即同发育过程相关.     

大鼠睾丸中S-100蛋白生后发育变化的免疫细胞化学研究

本文应用免疫细胞方法,研究大鼠生后４天、７天、１４天、３０天、２个月和３个月睾丸中Ｓ－１００蛋白的分布和变化规律。结果表明：Ｓ－１００蛋白染色反应位于睾丸间质细胞,直到生后３０天才出现阳性细胞,数量少,着色浅,而２月龄和３月龄大鼠睾丸Ｓ－１００蛋白阳性细胞数量多,着色深。提示Ｓ－１００蛋白可能参与间质细胞的合成和分泌睾酮过程。     

METABOLISM OF HIGHLY BASIC PROTEINS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— (1) The encephalitogenic basic protein obtained from adult rat brain by treatment with 0·03 N-HCl was demonstrable in the brain on the 10th day after birth. It showed a marked increase in quantity during the phase of active myelination.
(2) The proteins extracted under similar conditions from 5-day old rat brain contained several highly basic proteins other than the encephalitogenic basic protein. These basic proteins, which were electrophoretically similar to highly basic proteins extracted similarly from adult rat liver, are histones.
(3) For metabolic studies the entire group of highly basic proteins in the acid extract was obtained after one-step adsorption of other proteins on DEAE-cellulose equilibrated at pH 9·8
(4) After injection of [¹⁴C]lysine the fractions containing highly basic proteins, water soluble non-basic proteins and other tissue proteins of the brain showed higher relative specific radioactivities during the period 1–10 days after birth than during later stages of postnatal development. The fraction containing proteolipid protein, another myelin protein, showed a low relative specific radioactivity throughout the whole period of postnatal development. The relative specific radioactivity of proteolipid protein was somewhat higher in young than in adult rat brain.     

注射依赖cAMP的蛋白激酶催化亚基加速胚胎内细胞间连接通讯的发育(英文)

本研究以爪蟾胚胎内胚层细胞间连接通讯发育的时程为指标,观察了cAMP和依赖cAMP的蛋白激酶催化亚基对这一发育的影响。将依赖cAMP的蛋白激酶催化亚基注射入爪蟾胚胎四细胞期的每一个细胞可使该胚胎内胚层细胞间连接通讯的发育明显加快,提示在正常状态下这一发育的进程是受细胞内依赖cAMP的磷酸化水平的制约。但用双丁酰cAMP和磷酸二酯酶抑制剂对胚胎进行培育,或将cAMP和磷酸二酯酶抑制剂注射入胚胎细胞,对这一发育并无影响,可能是由于这些胚胎细胞内依赖cAMP的蛋白激酶的实际有效含量较低而cAMP含量较高所致。注射这种蛋白激酶催化亚基所诱导的胚胎细胞间连接通讯在注射后约8.5小时出现。这一时间比前人在哺乳动物细胞培养中用cAMP或必需的信使RNA诱导出细胞间连接通讯所需要的时间(2—4小时)长得多,提示在胚胎中依赖cAMP的磷酸化对细胞间连接通讯发育的作用是从RNA转录的水平上开始的。     

MEMBRANE PROTEINS OF RAT BRAIN: COMPOSITIONAL CHANGES DURING POSTNATAL DEVELOPMENT

Abstract— Membrane fractions from forebrain of rat were isolated at ages ranging from 5 to 93 days. Among these fractions were total membranes, three fractions isolated by density gradient centrifugation, and three subfractions which consisted of purified myelin and of two supernatant fractions. All membrane fractions showed an increase in protein content during the first postnatal month; however, only the myelin fraction and one of its supernatant fractions showed a prolonged accumulation. Myelin protein increased continually from 0.17 mg/g brain at 15 days to 8.3 mg/g brain at 93 days.
All fractions were analysed for protein composition by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Characteristic changes in protein composition were noted during postnatal development, most of which were pronounced up to the age of 20 days. Among others was a decrease in histones as compared to other proteins, with a concomitant shift in preponderance from the slow- to the fast-migrating histone band. In parallel, other proteins of high molecular weight became more prominent. No myelin could be isolated at 5 and 10 days. The deposition of myelin proteins was parallelled by the appearance of the Wolfgram protein which points to a close correlation of the Wolfgram protein to the process of myelination.     

MODIFICATIONS IN SOLUBLE PROTEIN KINASE AND CYCLIC-AMP BINDING CAPACITY OF DEVELOPING RAT BRAIN

Histone and casein phosphoprotein-kinase activities were determined in rat brain soluble fraction at various stages of development. Cyclic AMP -independent or basal histone kinase activity increased, whereas cyclic AMP -dependent activity decreased in whole soluble fraction with the age. On the contrary, whole soluble cyclic AMP -dependent and -independent casein kinases activities did not show any difference during development. The percentage of activation by cyclic AMP of histone kinase activity and [³H] cyclic- AMP binding activity in the soluble fraction decreased markedly during development. By DEAE-cellulose chromatography the histone kinase was separated mainly into 4 peaks; the fourth peak was strongly stimulated by cyclic AMP . Stimulation by cyclic AMP was higher in the 4-day-old rat brains than in the 9- and 30-day-old. In the 9-day-old rats the ratio of cyclic AMP -dependent histone kinase in respect to the cyclic AMP -independent enzyme was higher than in 4- and 30-day-old rats. Casein kinase activities in the brains of 9- and 30-day-old rats were separated by DEAE-cellulose chromatography into three peaks of which the third one was stimulated by cyclic AMP . Little, if any, difference was observed for casein kinase during the development. These results suggest that brain histone and casein kinase are different enzymes:     

胚胎及生后不同发育时期大鼠睾丸生殖细胞的凋亡

目的 探索雄性生殖细胞在发育过程中凋亡的特征和规律。方法 利用改进的石蜡切片原位末端标记法（TUNEL法）观察SD大鼠睾丸生殖细胞,对胚胎及生后不同阶段生殖细胞凋亡进行研究。结果 胚胎第13.5天原始生殖细胞即有较高的凋亡率,胚胎第19.5天到出生后第1天,未检测到凋亡生殖细胞,出生后第7天精原细胞分裂增生,伴有较高的凋亡率,与其他各年龄组有显著性差异。出生后第14天精母细胞凋亡率最高,与其他日龄组有显著性差异。结论 SD大鼠雄性生殖细胞发生,发育,成熟过程中都存在凋亡,主要发生在处于细胞增殖过程中的原始生殖细胞,精原细胞和初级精母细胞。     

THE POSTNATAL DEVELOPMENT OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Abstract— The postnatal development of three enzymes in the rat forebrain was studied. When expressed per tissue weight the catechol- O -methyl transferase (COMT) increased 2-fold from birth to adult age, the lactate dehydrogenase (LDH) 4-fold and the monoamine oxidase (MAO) 12-fold. Expressed per mg protein the increase in the enzyme activities in the subcellular fractions which contained the main part of the different enzymes was still 2–4-fold for COMT and LDH while for MAO it was 4-fold.
There was a relative increase in the COMT activity in the P₂ fraction (synaptosomes and mitochondria). This increase was identical with a corresponding increase in LDH activity and protein and was probably due to growth of nerve terminals. The COMT in the cytoplasm of the synaptosomes showed the same increase relative to the proteins as did the 'free' cytoplasmic enzyme.
The conclusion is drawn that the enzymes in the rat brain show a certain degree of development during brain growth. An additional increase of some enzymes is due to the development of specialized structures such as mitochondria and nerve terminals with synapses. COMT is not related to any such specialized structure.     

THE DESMOSTEROL REDUCTASE ACTIVITY OF RAT BRAIN DURING DEVELOPMENT1

Abstract— The reduction of desmosterol by cell-free preparations from developing rat brain was established and the age-dependent alterations in reductase activity were correlated with levels of desmosterol in brain. An increase in enzymic activity followed closely the sharp increase in levels of desmosterol that was observed at about 5 days of age and that reached a maximum at 8-11 days of postnatal age. Furthermore, the abrupt decrease in the desmosterol content of brain at 13-15 days of age was associated with a decrease in enzymic activity. We suggest that the enzyme may be substrate-induced and that this attribute may be of significance with respect to the ontogenesis of myelin. Cerebral desmosterol reductase exhibited a specific requirement for NADPH and was primarily a particulate enzyme.     

吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The K_m for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K_m for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K_m for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.     

EFFECTS OF HYPOTHYROIDISM AND UNDERNUTRITION ON DNA CONTENT AND THYMIDINE KINASE ACTIVITY DURING CEREBELLAR DEVELOPMENT IN THE RAT1,2

—The effects of hypothyroidism and several degrees of undernutrition on the development of cerebellar weight, DNA, and thymidine kinase activity were studied in young rats ranging in age from 2 to 22 days. Early propylthiouracil treatment caused a delayed cerebellar cell multiplication. The activity of cerebellar thymidine kinase was suppressed at ages 2 and 5 days and was in excess of control values on days 15 and 22, thus resulting in a delay in the developmental spectrum for thymidine kinase, and extending the time span of activity beyond that of controls. Undernutrition led to varying degrees of reduced cell proliferation at experimental ages 5, 12, and 19 days. Cerebella from the most undernourished animals showed significant differences from controls in thymidine kinase activity at ages 5 and 12 days. Comparisons between sub-groups from within the oversized litters at 5 and 12 days suggested that changes in thymidine kinase activity relate to the degree of undernutrition to which the sub-group is subjected and that during development there may be a critical degree of undernutrition at which a particular essential enzyme becomes affected. This study emphasizes the biochemical similarities and differences between neonatal hypothyroidism and undernutrition, while pointing out the difficulties which exist in biochemical separation of components of the two conditions. Further evidence is presented that thymidine kinase is responsive to hormonal stimuli during cerebellar development and may play an important role in the regulation of DNA biosynthesis in brain as well as other organs.     

PROTEIN PATTERNS IN DIFFERENT LOBES AND DURING DEVELOPMENT OF OCTOPUS BRAIN

Abstract— Utilizing techniques of continuous and SDS-electrophoresis we have examined the saline-soluble and SDS-soluble (membrane-bound) proteins extracted from the main lobes of adult octopus brain and from the developing optic lobe of the same species. Several additional protein bands are present among the soluble and the membrane-bound proteins of the vertical lobe in comparison with the suboesophageal lobe. Since the former contains an essentially homogeneous population of small neurons, while the suboesophageal lobe is rich in large nerve cells, these protein bands have been attributed to the small neuronal type present in the vertical lobe.
In the course of a 10,000-fold increment in body weight, from 0.4 g to 4 kg, there is a significant increase in the concentration of several soluble proteins extracted from the optic lobe. Three of these proteins increase to a marked degree. Among the membrane-bound proteins some show a moderate increase with age while other protein components of smaller molecular weight undergo a moderate decrease. The overall tissue concentration of the membrane-bound proteins increases between 0.4 g and 50 g body weight, slightly declining in animals of larger size.