A COMPARATIVE STUDY OF THE ISOLATION OF THE CORTEX AND THE ROLE OF THE CALCIUM-INSOLUBLE PROTEIN IN SEVERAL SPECIES OF SEA URCHIN EGG

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.     

PURIFICATION AND CHARACTERISTICS OF RNase INHIBITOR FROM PIG CEREBRAL CORTEX

Abstract— An RNase inhibitor has been purified from pig cerebral cortex by DEAE-cellulose and hydroxylapatite chromatography and Sephadex G-100 gel filtration. The purified RNase inhibitor could be resolved into a major band (about 80–85 per cent of total protein) and several minor components by polyacrylamide gel electrophoresis.
The ultraviolet absorption curve of the purified RNase inhibitor indicated a typical protein spectrum. The inhibitor was inactivated by digestion with trypsin or prozyme, and by heating at 70ºC for 5 min. The inhibitor was also inactivated by an SH reagent such as p -chloromercuribenzoate. The inhibitor did not affect RNase T₁. It has been suggested that the inhibitor is an acidic protein and also a SH-protein. The molecular weight of the RNase inhibitor was estimated to be about 60,000.     

Macrophage Activation by Tetrahymena pyriformis II. Active Protein Fractions from Tetrahymena

ABSTRACT. Water-soluble and 0.6 M KCl-soluble protein fractions prepared from Tetrahymena pyriformis , when inoculated into mice, could effectively induce activated macrophages having the ability to kill Toxoplasma gondii in vitro. This effect was not induced by other proteins tested, such as bovine serum albumin, pepsin from porcine stomach mucosa and chicken egg-white lysozyme, nor by muramyl dipeptide (MDP), a potent immunoadjuvant. Five fractions obtained by DEAE-Sephadex chromatography of the water-soluble protein fraction were compared with regard to induction of toxoplasmacidal activity in macrophages. The first peak was most effective for activation of macrophages. Five fractions obtained by chromatography of the 0.6 M KCl-soluble protein fraction were also examined and it was found that the first peak had the activity. No marked difference in activity was observed between the active fractions of water-soluble and 0.6 M KCl-soluble protein fractions. For practical use, we focused on the water-soluble active fraction. The minimum effective dose of the active fraction was 100 μg and the fraction could activate macrophages directly in vitro. Four fractions obtained by gel filtration of the active fraction on Sephadex G-200 were compared and the first peak had the activity. The first peak contained a single protein, revealed by SDS-polyacrylamide gel electrophoresis; its apparent molecular weight was 64,000.     

SEPARATION OF ACETYLCHOLINE AND CATECHOLAMINE CONTAINING SYNAPTIC VESICLES FROM BRAIN CORTEX

Abstract— Synaptic vesicles were prepared from guinea-pig cerebral cortex on a continuous D₂O-H₂O(1:1)-sucrose gradient and purified in the presence of 1 m m -EGTA by chromatography on columns of glass beads of controlled pore size. As markers, endogenous ACh, NA, dopamine and DβH were measured.
Two distinct populations of synaptic vesicles were recognized between the layers of 0.2–0.3 m - and 0.3–0.5 m -sucrose, which differed from each other both in electron microscopic appearance and transmitter content. The less dense vesicles had a much higher ACh content than the more dense vesicles which were composed mainly of somewhat larger particles with high NA and dopamine content. DβH was found to be present in substantial amounts in guinea-pig cortex and was located in the synaptic vesicle fractions having high CA content.
After glass bead chromatography the vesicle preparations were morphologically homogeneous, practically free from other subcellular elements and were contaminated with each other by not more than 10%
The yields were 0.2 and 0.1 mg protein g cortex⁻¹ tissue for 'cholinergic' and 'adrenergic' vesicle preparations, respectively.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: l-ASPARTIC ACID-BINDING PROTEIN FROM THE RAT CEREBRAL CORTEX

Abstract— Lyophilized rat cerebral cortex was treated with chloroform-methanol (2:1, v/v), and the extracted hydrophobic proteins (i.e. proteolipids) were separated by column chromatography on Sephadex LH-20. The first peak of protein, eluting with chloroform in the void volume, had high affinity binding for l -[¹⁴C]aspartic acid. The saturation of the binding showed three saturable sites with apparent dissociation constants of 0.2 μ m , 10 μ m and 50 μ m . The binding capacities of the three sites were 2.8, 132 and 617 nmol/mg of protein, respectively. There were 8.0 nmol of high affinity binding sites for l -aspartic acid and 1.53 nmol for l -glutamic acid per g of fresh tissue in the cerebral cortex of the rat. Differentiation between binding of l -aspartic and l -glutamic acid was clearly established by cross-binding and competition experiments with agonists and antagonists.
It is suggested that the isolated protein fraction may correspond to a synaptic receptor and not to the transport system. It is concluded that in the cerebral cortex there is a separate receptor for l -aspartic acid. This is further support to the possible role of this amino acid as a central excitatory transmitter.     

DEMONSTRATION OF HIGH AFFINITY HEXOSE UPTAKE IN CEREBRAL CORTEX SLICES

Abstract— The high-affinity glucose transport system ( K _m 0.2–0.4 m m ), previously detected in synaptosome preparations, has now been demonstrated to be present in slices of the cerebral cortex incubated in vitro. The kinetic properties of this undirectional uptake process in slices were similar to those exhibited by synaptosomes. The results are discussed with respect to the possible sites of the high affinity and low affinity glucose transport processes in the brain.     

SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA-PIG CEREBRAL CORTEX AND SPLEEN

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea-pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non-occluded micro-particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [³H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non-occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6-0.8 m -sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub-fractions D to G (0.4-1.0 m -sucrose) and the other in sub-fraction J (1.4 m -sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.
Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4-0.6 m -sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m -sucrose).     

EFFECTS OF ANOXIA IN VITRO ON CELLULAR RESPIRATION OF BRAIN CORTEX

Abstract— –Anaerobic preincubation of slices of brain cortex from adult rats in saline at 37°C, but not at 20°C, impaired their capacity to oxidize glucose and other substrates. This effect was to a large extent prevented by including either glucose + oxaloacetate, or l -glutamine + l -aspartate +α-oxoglutarate in the medium during anaerobic preincubation, and in addition the anaerobic uptake of water by the tissue was decreased. The respiratory mechanism in slices of brain cortex from 1-day-old rats was much less affected by anoxia and sensitivity to anoxia develops with age.     

SUB-CELLULAR LOCALISATION OF ENHANCED LYSINE INCORPORATION INTO CEREBRAL CORTEX PROTEINS IN DARK-REARED AND LIGHT-EXPOSED RATS

Incorporation of lysine into acid-insoluble material from subcellular fractions of rat cerebral cortex has been studied using double and single-labelling techniques, in littermates reared for 50 days in the dark and then dark-maintained or light-exposed for 1 h. When light-exposed animals were compared to dark controls the only subcellular fraction from the whole cortex in which lysine incorporation shows a significant elevation (168%, P < 0.05) was located in the ribosomal pellet of the cerebral cortex. A similar comparison of subcellular fractions from visual and motor cortices showed that the elevation was again in the ribosomes and confined to visual cortex only. Motor cortex of light-exposed animals showed a small depression of incorporation in ribosomes as compared to dark controls. Sub-fractionation of nuclei from whole cortex preparations showed varying, but non-significant elevations in light-exposed animals in all but the histone fraction in which there was negligible incorporation of precursor. It is concluded that enhancement of incorporation of precursor into proteins of the cerebral cortex, which accompanies first exposure to light, is a complex response. At exposure for 1 h it involves a number of particular protein species located in the visual cortex, a major proportion of which are ribosomally bound.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

SOME PROPERTIES OF THE PROTEIN FORMING THE OUTER FIBERS OF CILIA

Cilia were isolated from Tetrahymena pyriformis by an ethanol-calcium procedure. Solutions of outer-fiber protein were obtained either by aqueous extraction of an acetone powder of whole cilia, or by dissolving the isolated outer-fibers in 0.6 M KCl. In aqueous solution, the outer-fiber protein has a sedimentation coefficient of 6.0S and a molecular weight of 104,000 ± 14,000. In 5 M guanidine hydrochloride solution the molecular weight falls to 55,000 ± 5,000. After reduction and alkylation in 8 M urea, about 95% of the protein migrates as a single band on electrophoresis in polyacrylamide gel at pH 8.9; the migration velocity is identical with that of reduced and alkylated actin. Freshly prepared outer-fiber protein contains about 7.5 sulfhydryl groups per 55,000 g of protein. The amino acid composition of outer-fiber protein resembles that of actin, with such differences as occur being of the same order as those between actins from different species of animal.     

COMPARISON OF THE EFFECTS OF DEPOLARIZING AGENTS AND NEUROTRANSMITTERS ON REGIONAL CNS CYCLIC GMP LEVELS IN VARIOUS''ANIMALS

Abstract— The effects of 121 m m -K⁺, 10 m m -glutamate, 5 m m -GABA, 1 m m -glycine, 0.1 m m -NE, and 1–10 μ m ACh on cyclic GMP levels in tissue slices prepared from cerebral cortex and cerebellum of mouse, rabbit, guinea-pig, cat, and rat were studied. Basal levels of cyclic GMP in the cerebella of mice, guinea-pigs and cats were 4–15 and 70 pmol/mg prot in rat, whereas in the cerebral cortex of the same animals, levels were only 0.6–2 pmol/mg prot. In contrast, basal levels of the cyclic nucleotide were 1–2 pmol/mg prot in both of these regions in rabbit brain. Only 121 m m -K⁺ was capable of increasing cyclic GMP levels in all the tissues studied. Elevations ranged from 30% in rat cerebral cortex to 2800% in mouse cerebellum. Glutamate produced a 30–1000% rise of cyclic GMP levels in all tissues except rabbit cerebellum. NE elevated levels of cyclic nucleotide 2- to 3-fold in slices of cerebellum from all species studied but had no effect in cerebral cortex. GABA and glycine had no effect in any tissue except mouse cerebellum. ACh had no consistent effect on levels of cyclic GMP in any brain region investigated. These results suggest that mechanisms regulating cyclic GMP levels in mammalian CNS vary among brain regions and among animal species.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

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.     

DIFFERENTIAL INCORPORATION OF [3H]LYSINE INTO VISUAL CORTEX PROTEIN FRACTIONS DURING FIRST EXPOSURE TO LIGHT

—A resolution of the enhancement of protein synthesis in the visual cortex of rats during first exposure to light (Richardson and Rose , 1972) was achieved by polyacrylamide gel electrophoresis using a double-labelling technique. Differential incorporation of lysine was established between exposed and control animals in two fractions of the soluble proteins and seven fractions of the insoluble proteins. This suggests that exposure to a new experience of this type involves a specific effect on protein synthesis, rather than a general stimulation across all fractions.     

急性给锂小鼠大脑皮层NOS活性与蛋白表达的时程变化

目的探讨急性给锂小鼠大脑皮层一氧化氮合酶(NOS)活性与蛋白表达的时程变化及其意义.方法选用昆明小鼠40只,分为对照组和腹腔注射1.5mmol/Kg氯化锂(LiCl)即刻、0.5h、1h、3h、6h、12h、24h组,每组5只.采用NADPH-d黄递酶组织化学和ABC免疫组化法,观察急性给锂后不同时程小鼠大脑皮层NOS和nNOS阳性神经元数目的变化.结果急性给锂即刻小鼠大脑皮层NOS和nNOS阳性神经元数目明显增加(P＜0.01),1h后达到高峰(P＜0.01),6h和12h恢复到正常水平(P＞0.05),24hNOS阳性神经元又明显增高(P＜0.01),nNOS阳性神经元处于正常水平(P＞0.05).结论本实验提示急性给锂对小鼠大脑皮层NOS和nNOS阳性神经元数目有一定影响,这种变化可能是锂影响脑发育及锂的神经毒性的机理之一.     

STUDIES ON SULFHYDRYL GROUPS DURING CELL DIVISION OF SEA URCHIN EGG : III. —SH Groups of KCl-Soluble Proteins and Their Change during Cleavage

Sea urchin egg proteins extracted with KCl are mostly TCA-soluble and, conversely, those extracted with TCA are KCl-soluble. Both groups are water-insoluble and show fluctuations in—SH content during the division cycle. The fluctuation of the—SH groups of the KCl-soluble protein of the whole egg is due to a —SH—S—S— interchange within the freely reacting groups and not within the sluggish and masked —SH groups of the protein. The —SH content of the KCl-soluble protein of the egg cortex also fluctuates in a similar way.     

DEVELOPMENT AND CHARACTERISTICS OF THE HISTAMINE-INDUCED ACCUMULATION OF CYCLIC AMP IN THE RABBIT CEREBRAL CORTEX

Abstract— In slices of adult rabbit cerebral cortex histamine at 5 μM produced a detectable rise in adenosine 3',5'-monophosphate (cyclic AMP). A maximum (20-fold) increase was observed in response to 0–5 mM histamine, with higher concentrations being less effective. The antihistaminic agent, tripelennamine, inhibited the response to 50 μM histamine in a dose-related manner. No effect on basal levels of cyclic AMP was noted with the highest dose of tripelennamine. The cyclic AMP response to 50 μM histamine was sustained for up to 1 h of incubation whether the slices and included medium were assayed together or the slices were assayed separately, although after 60 min of incubation cyclic AMP levels were higher when the medium was included in the assay. During development of the rabbit cerebral cortex, the first detectable increase of cyclic AMP in response to histamine occurred at fetal day 25, and from day 28 to birth the response was a 4-to 5-fold increase. A maximal (10-fold) response was observed at 4–8 days postpartum and by 20 days of postnatal age the response had decreased to the adult levels.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

免疫印迹技术的一些改进

本文描述了作者在研究低等生物着丝粒蛋白过程中对免疫印迹技术所作的一些改进：１．分离胶由１２％ＳＤＳ聚丙烯酰胺均一胶改作５％—１２％（或１５％）的线性梯度胶,使不同分子量范围的蛋白都能得以较好地分离;２．用眼虫色素代替溴酚蓝作示踪剂,以能适时地终止电泳;３．转移由４００ｍＡ３．５ｈ改为２００ｍＡ１７ｈ,由冰箱内空气冷却改为流水浴冷却,提高了冷却效率;４．对转移后的硝酸纤维素膜作除ＳＤＳ处理;使转移后的蛋白在一定程度上得以复性;５．抗体孵育温度由３７℃或室温１—２ｈ改为４℃冰箱过夜,降低了本底,提高了印迹反应的特异性。我们已用改进后的方法对一些低等生物作了一系列的着丝粒蛋白研究,取得了比较满意的结果。