PARTICULATE AND SOLUBILIZED FUCOSYL TRANSFERASES FROM MOUSE BRAIN

The transfer of [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to endogenous and exogenous acceptors by particulate and solubilized preparations from mouse brain is described. Suspensions of brain microsomes incorporated [¹⁴C]fucose into a heterogenous group of glycoprotein products, which have a distribution on gel electrophoresis similar to those synthesized in vivo. Fucosyl transferase, extracted from brain microsomes by Triton X-100, transferred [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to terminal galactose residues exposed by mild acid hydrolysis of porcine plasma glycoprotein. Comparison of the specific activities of the solubilized fucosyl transferase from a number of organs showed that, in the presence of the exogenous acceptor which was used, the transferase of brain was more active than the transferases from all other organs tested, with the exception of kidney. Examination of subcellular fractions of brain, with endogenous and exogenous acceptors, showed that activity was limited to fractions containing microsomal membranes, whereas synaptosomal and other fractions were virtually inactive.     

DIFFERENTIATION OF NEURONAL TYPES AND SYNAPSES IN MYELINATING CULTURES OF MOUSE CEREBELLUM

The Holmes silver impregnation method has made possible the recognition of multiple neuronal types and synapses in myelinating cultures of mouse cerebellum. Well stained large and medium-sized neurons are always found in small numbers near ependymal formations and are considered to be roof nuclear neurons. Neurons with poorly stained somas, abruptly demarked from intensely stained axons, are numerous and often are arranged in palisades. With prolonged maintenance in vitro these neurons develop some but not all of the features of mature Purkinje cells. A few small, densely stained, bipolar neurons, often with one process bifurcated, are found in dense regions of some cultures of newborn cerebellum. These neurons are commoner in cultures from cerebella of older mice. They closely resemble the immature granule cell in vivo. All the neuron types recognized in cultures are present in the initial explants; neurons differentiate further in vitro, but new neurons probably do not form. Synaptic boutons are found on somas and dendrites of many Purkinje cells. Two cultures contained structures resembling the basket endings which surround Purkinje cell somas in vivo. The complexity of neuronal relationships in cultures of central nervous tissue is emphasized.     

ELECTRON MICROSCOPIC OBSERVATIONS OF RAT AND MOUSE CEREBELLUM IN TISSUE CULTURE

Closely ordered stages of myelin formation in cultures of newborn rat and mouse cerebellum, selected by direct light microscopy, were studied with the electron microscope. Electron micrographs of these cultures reveal the presence of neurons, axons, neuroglia, microglia, and ependymal cells. The appearance of the neuron is identical to that previously described in vivo. The neuroglial cell has long, branching processes, and its cytoplasm is characterized by packets of long, narrow fibrils. During myelin formation, a glial cell process surrounds the axon. This process may form an internal mesaxon and may spiral for several turns around the axon. Other glial cell processes may interdigitate with or overlay the innermost process to contribute to the multilamellated structure. The glial processes flatten and the cytoplasmic surfaces of the cell membrane come into contact to form the lamellae of the myelin sheath. These adhesions may be temporarily incomplete as evidenced by sequestered islands of glial cytoplasm among the myelin lamellae. Ultimately, a compact, apparently spiral, myelin sheath is formed. These findings are discussed in relation to in vivo central myelin formation.     

烟色红曲霉耐热解脂酶的形成及特性

烟色红曲霉(Monacus fulginosus)M-101菌株经麦麸固态培养生成耐热脂肪酶和酯酶。产酶的适宜条件为:培养温度30℃,初始pH 3.0—3.5,麸曲初始含水量75％。培养4—5天后,脂肪酶活力可达207u/g,酯酶活力达14.6u/g。粗酶试验表明,脂肪酶和酯酶的最适反应温度为50℃,脂肪酶最适反应pH为6.0。酯酶最适反应pH为6.8。酯酶耐热性略高于脂肪酶,在55℃处理1小时和45℃处理24小时,两种酶活力基本不变。     

提前接受光照对小鼠视网膜TH和bFGF表达的影响

目的在通过手术使小鼠提前睁眼接受光照,并成功诱发近视的基础上,进一步研究TH和bFGF在视网膜中的表达变化,以探讨早产近视的发病机理.方法实验用新生第4d的C57BL/6J小鼠,通过手术分离单侧上、下眼睑,使之提前睁眼并接受正常光照,在第14d检查两眼屈光力,应用RT-PCR和免疫组化方法,检测视网膜中bFGF和TH的表达.结果提前接受光照能诱导形成-9.77±0.09D的相对近视.RT-PCR结果显示提前光照小鼠视网膜TH和bFGFmRNA含量明显减少(t值分别为4.316和12.189,P<0.01).免疫组织化学显示TH免疫阳性反应主要分布在节细胞层、内网层和内核层,bFGF免疫阳性反应主要分布在内网层及其两侧的部分节细胞和内核层细胞,另在内网层和视锥视杆层也有微弱表达.免疫组织化学染色显示提前光照小鼠视网膜中TH和bFGF的表达量明显下降.结论研究表明新生小鼠提前光照后,其视网膜TH和bFGF的表达都明显下降,提示TH和bFGF与早产近视的形成有密切关系.     

NEUROCHEMICAL ASPECTS OF NEURONAL ONTOGENESIS IN THE DEVELOPING RAT CEREBELLUM: CHANGES IN NEUROTRANSMITTER AND POLYAMINE SYNTHESIZING ENZYMES

Abstract— Changes in the activities of several specific enzymes were measured in the cerebellum during development. Early transient increases were found in both ornithine decarboxylase and S -adenosylmethionine decarboxylase, enzymes involved in the initial steps of polyamine synthesis. Different patterns of changes were found in neurotransmitter synthesizing enzymes. Tyrosine hydroxylase activity achieved adult levels very early, by 3 days after birth, and remained at this level. Glutamic acid decarboxylase activity, while very low at early stages, increased rapidly before birth and then after a lag period of 10 days started to increase rapidly, directly related to the general growth of cerebellar weight and protein content. Choline acetyltransferase activity started to increase rapidly, reaching a peak of about 100% of adult levels at 3-7 days after birth; the activity then gradually declined and at 20 days, after reaching a low of about 55% of adult values, gradually started to increase, reaching adult levels later than 40 days after birth. The development of protein carboxymethylase activity was similar to that of glutamic acid decarboxylase, directly related to the general growth of the cerebellum. Several interpretations of the results are discussed.     

ENZYMATIC BIOSYNTHESIS OF ETHANOLAMINE- AND CHOLINE-PHOSPHOGLYCERIDES IN RETINA DURING DEVELOPMENT. EFFECT OF LIGHT STIMULATION

Choline- and ethanolamine-phosphoglycerides (CPG and EPG) are the most abundant phospholipids of retinal membranes. We have investigated some regulatory mechanisms involved in the final steps of their biosynthesis, namely those catalysed by CDP-choline 1,2 diradyl-sn-glycerol choline phosphotransferase (CPT) and CDP-ethanolamine 1,2 diradyl-sn-glycerol ethanolamine phosphotransferase (EPT). We have studied both enzymes in the retina which offers an excellent model for the investigation of the molecular basis of the effect of its physiological stimulus, the light. In chick retina. the specific activity (SA) of EPT reached a maximum at the 18th day of embryonic life and decreased thereafter. In the case of CPT, a similar peak of SA was observed at hatching. The time of maximum SA of EPT and CPT corresponded to the period during which retinal rod outer segments are formed. The apparent K_m values of EPT and CPT determined with whole retinal homogenates for CDP-bases showed different profiles. The apparent K_m of EPT decreased during embryonic life and increased thereafter whereas the apparent K_m of CPT did not change during ontogenesis. Light stimulation of calf retinal homogenates had different effects on phosphotransferase activities. In the presence of only endogenous diacylglycerol (DAG) the SA of CPT was 2-fold higher for dark-adapted retinas, whereas no differences in EPT activities were observed. After addition of exogenous DAG (4mM) to the incubation medium, light stimulation of the retina led to a 50% increase of EPT activity whereas no effect was observed for CPT. These different effects could be related to the cyclic nucleotides present in retina before and after light stimulation. In addition all the data presented in this study indicate that, as in brain, CPT and EPT in retina are two different enzymes.     

CYCLIC NUCLEOTIDE LEVELS IN NORMAL AND BIOLOGICALLY FRACTIONATED MOUSE RETINA: EFFECTS OF LIGHT AND DARK ADAPTATION

Abstract— The content of cyclic AMP and cyclic GMP was measured in whole eyes and in normal retinas from C57BL(6)J mice, in receptorless retinas from congenic mice homozygous for the receptor dystrophy gene (rd/rd), and in retinas from mice treated postnatally with monosodium glutamate. Normal retinas contain approx 320 μg of protein: dystrophic (rd/rd) retinas contain approx 110μg of protein, lack rods but possess some surviving cone somata and terminals: glutamate-modified retinas contain approx 200 μg of protein and have both a reduced area and thickness with a marked deficiency of ganglion cells and amacrine cells. In normal mice, more than 90% of the cyclic GMP, but only 607, of the cyclic AMP of the whole eye was in the retina. In normal dark-adapted retinas isolated under dim red light cyclic AMP and cyclic GMP content was 4.1 and 20.2pmol/retina, respectively. The content of both cyclic AMP and cyclic GMP was 40% less, 2.5 and 11.5pmol/retina, respectively, in light-adapted retinas. In dark-adapted retinas isolated under infra-red light, cyclic AMP content was 40%, higher than that in retinas isolated under dim red light; cyclic GMP content was the same under these two conditions. Receptorless retinas contained approx 50% as much cyclic AMP and only 1-2% as much cyclic GMP as normal retinas. Although glutamate-modified retinas also had approx 50% as much cyclic AMP, they contained 60-85%, as much cyclic GMP as normal retinas. Light decreased by 30-50% levels of both cyclic AMP and cyclic GMP in glutamate-modified retinas, but only reduced cyclic nucleotide levels in receptorless retinas by 20%.
These data indicate that 95% or more of the cyclic GMP is in photoreceptor cells, whereas cyclic AMP is more evenly distributed throughout the retina. In addition, both cyclic AMP and cyclic GMP levels are influenced by light- and dark-adaptation.     

SOLUBLE AND MEMBRANE BOUND CARBONIC ANHYDRASES FROM RAT CNS: REGIONAL DEVELOPMENT

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.     

DISTRIBUTION OF FOLIC ACID COENZYMES AND FOLATE DEPENDENT ENZYMES IN MOUSE BRAIN1

—Folic acid coenzymes were found to be distributed equally between post-nuclear particulate and soluble fractions from whole Swiss mouse brain. Mitochondria isolated from the particulate fraction contained essentially only the N⁵-methyl derivative of folate, virtually all of which was in a polyglutamate form. Isolated synaptosomes contained significantly more folate than did mitochondria, with the greater proportion being non-N⁵-methyl derivatives. Osmotic lysis of synaptosomes released only a small portion of the folate; approximately 80 per cent remained with the particulate components of the synaptosome. The enzymes serine transhydroxymethylase and N⁵, N¹⁰-methylenetetrahydrofolate dehydrogenase were found in both the soluble and particulate fractions while formiminoglutamic acid:tetrahydrofolate formiminotransferase activity could not be detected. These findings may be of importance with respect to the synaptic functions of folate coenzymes, including the methylation of biogenic amines.     

ELECTROPHORETIC ANALYSIS OF SOLUBLE ENZYMES IN FIVE FRESHWATER DINOFLAGELLATE SPECIES

Electrophoretic analysis of 11 different clonal freshwater Peridinium isolates shows that currently used morphological taxonomic criteria are conservative measures of relatedness and that the presence or absence of an apical pore is an important taxonomic feature. Correlation of malate dehydrogenase and glutamate dehydrogenase isoenzyme patterns with chromosome number variations among our isolates lends no support to polyploidy as a speciation mechanism.     

真鲷视网膜结构及其视觉特性研究：Ⅱ视网膜光感受细胞超微结构研究

对真鲷光感受细胞的超微结构进行观察,结果表明：视杆外段膜盘为游离膜盘,视锥外段膜盘则为连续的膜结构,视锥和视杆均含有连接纤毛和辅助外段。花萼状突起起源于内段。椭体内充满线粒体,无球状小体。双锥椭圆体并生膜为六层,视锥内段无鳍状突起,视锥突触带,在明适应视网膜中数量增多,在暗适应视网中数量减少,视杆突触带在这两种适应网膜中数量不变,每一杆小球只有一个突触带,而锥小足有４－６个突触带。     

THE TOXIC EFFECT OF SODIUM GLUTAMATE ON RAT RETINA: CHANGES IN PUTATIVE TRANSMITTERS AND THEIR CORRESPONDING ENZYMES

Abstract– Subcutaneous administration of high doses of sodium glutamate to rats during their first week after birth produced an almost total loss of choline acetyltransferase, a 90% reduction in glutamate decarboxylase and 70% reductions in acetylcholinesterase and DOPA decarboxylase activities in the adult retina. In addition there was a 70% decrease in GABA and 35-55% decrease in aspartate, glutamate, glycine, alanine and glutamine. No reduction in taurine was observed. The results support the view that the enzymes are mainly localized in the interneurons of retina and that taurine is present in the photoreceptor cells.
Glutamate treatment was also followed by a small reduction in choline acetyltransferase and glutamate decarboxylase of the superior colliculus and in choline acetyltransferase of hippocampus, whereas no changes could be detected in the lateral geniculate body of the adult rat. Unilateral enucleation performed on 1-day-old animals did not alter choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase and DOPA decarboxylase activities in the superior colliculus and in the lateral geniculate body of the adult rat.     

PHOSPHORYLATION OF NUCLEAR PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER IN VITRO

Abstract— Phosphorylation of nuclear protein was investigated with isolated nuclei from rabbit cerebral cortex, cerebellum and liver by using [γ-³²P]ATP. The results were compared with the previously reported findings on phosphorylation with tissue slices and [³²P]phosphate. Cerebral cortex showed a very high level of phosphorylation, while liver showed the lowest, the difference being several fold in magnitude. With each tissue source, the extent of phosphorylation was maximum at incubation period for 2–3 min with steady decline afterwards. When nuclear proteins were further fractionated into 0.14 m -NaCl-soluble, 0.25 n -HCl-soluble (mainly histone) and acidic phenol-soluble proteins, NaCl-soluble protein showed the highest phosphorylation while HCl-soluble the lowest. The ratio among these tissue sources studied and the ratio among various protein fractions in each tissue source were strikingly similar to what had been shown with tissue slices. Further separation of acidic phenol-soluble protein with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed retention of the characteristic difference of the pattern of phosphorylation between liver and the CNS tissue as having been observed with tissue slices, although phosphorylation of proteins with molecular weights of less than 40,000 was much reduced with the isolated nuclei. Although other methods with extracted protein kinase or chromatic protein fractions might be more desirable under ordinary situations, the system for nuclear protein phosphorylation with isolated nuclei and [γ-³²P]ATP may be useful under certain experimental conditions provided the incubation condition is carefully selected.     

KINETICS OF ROD OUTER SEGMENT RENEWAL IN THE DEVELOPING MOUSE RETINA

The kinetics of rod outer segment renewal in the developing retina have been investigated in C57BL/6J mice. Litters of mice were injected with [³H]amino acids at various ages and killed at progressively later time intervals. Plastic 1.5 µm sections of retina were studied by light microscope autoradiography. The rate of outer segment disk synthesis, as judged by labeled disk displacement away from the site of synthesis, is slightly greater than the adult level at 11–13 days of age; it rises to more than 1.6 times the adult rate between days 13 and 17, after which it falls to the adult level at 21–25 days. The rate of disk disposal, as measured by labeled disk movement toward the site of disposal, is less than 15% of the adult level at 11–13 days of age; it rises sharply to almost 70% of the adult level by days 13–15 and then more gradually approaches the adult rate. The net difference in rates of synthesis and disposal accounts for the rapid elongation of rod outer segments in the mouse between days 11 and 17 and the subsequent, more gradual elongation to the adult equilibrium length reached between days 19 and 25. The changing rate of outer segment disk synthesis characterizes the late stages of cytodifferentiation of the rod photoreceptor cells.     

CHARACTERISTICS AND PRODUCTS OF A CELL-FREE POLYPEPTIDE SYNTHESIZING SYSTEM FROM NEONATAL AND ADULT MOUSE BRAIN

—The regulation of protein synthesis by ribosomes isolated from mouse brain tissue was studied using a cell-free polyphenylalanine synthesizing system. Polypeptide synthesis was followed by assaying translocation and analysing the reaction products by BD-cellulose chromatography. The brain ribosomal activity could be divided by these methods into two distinct steps : binding of aminoacyl-tRNA to the ribosome and active translocation leading to subsequent polyphenylalanine synthesis. In comparison to initial binding of aminoacyl-tRNA, translocation in the cell-free system increased the incorporation of labelled phenylalanine by 10-fold. An analysis of the reaction products clearly showed active ribosomal synthesis of oligophenylalanine from [³H]phe-tRNA. Ribosomes isolated from neonatal brain tissue were 2–4 times as active as those obtained from adult brain tissue in polypeptide synthesis. In addition, polypeptides synthesized on the more active ribosomes from neonates tended to be of greater chain length than those from adult. Therefore, the maturation-dependent decrease in ribosomal protein synthetic activity during neural development was shown to be directly associated with the ribosome particles.     

STRUCTURE-LINKED ACTIVITY OF LYSOSOMAL ENZYMES IN THE DEVELOPING MOUSE BRAIN

—A longitudinal study of the maturation of mouse cerebral lysosomal enzymes has been completed. Activity of the enzymes, acid phosphatase (I.U.B. 3.1.3.2), β-glucuronidase (I.U.B. 3.2.1.31) and β-acetylglucosaminidase (I.U.B. 3.2.1.30) was assayed spectrofluorimetrically on portions of supernatant from 0.25 M sucrose homogenates spun at 6 x 10³ -min. Activities were obtained in native (free) and Triton X-100 activated samples (total). The neonatal period was characterized by relatively low free and high total acid phosphatase activities. An abrupt rise in free activity occurred during the period 10–20 days. Discontinuous anion exchange DEAE cellulose chromatography (0.01 m -tris–maleate, pH 6.3) with elution by ascending molarities of NaCl of the Triton X-100 activated supernatant revealed three major peaks in the adult. A fourth peak, designated as fraction II (‘maturation fraction’) occurred only during the neonatal period, a time also characterized by increased specific activity of fraction I, with no change in fraction IV. The chromatographic fractions were further characterized by optimal pH, ascorbate, fluoride, Cu²⁺ and Fe²⁺ ions. The maturation profiles of total, β-glucuronidase and total, β-acetylglucosaminidase differed from each other, and from that of total acid phosphatase. Comparable differences existed in the profiles of the free activities, and the ratio of free:total activity differed for each enzyme at any selected time especially during the neonatal period. These findings are are discussed with reference to the maturation of isoenzyme fractions with age, and suggest that the changes in structure-linked organization of individual lysosomal hydrolases are functions of heterogeneity in enzyme complement of individual lysosomes.     

THE PERMEABILITY OF ISOLATED AND IN SITU MOUSE HEPATIC GAP JUNCTIONS STUDIED WITH ENZYMATIC TRACERS

We have studied the effects of phospholipase C from Clostridium welchii on gap junctions in the intact mouse liver and in a junction-rich fraction prepared from mouse liver. Treatment of the isolated junctions results in the disappearance of both the 20 A gap and of the polygonal lattice visible with lanthanum. The junctions are morphologically unaltered, however, when whole livers are perfused with phospholipase via the portal vein. These results suggest that extracellular phospholipase cannot diffuse into the junctional area, but that the enzyme may affect structures within the gap from its cytoplasmic surfaces which become exposed in the isolated preparations. Horseradish peroxidase, which has physical dimensions similar to those of Clostridium phospholipase is also denied access to the 20 A gap in whole liver, while peroxidase reaction product can be seen in the gap in isolated preparations. Beef liver catalase, however, a tracer molecule much larger than peroxidase, cannot penetrate even in isolated fractions. If the cytoplasmic approaches to the gap junction used by peroxidase and phospholipase are available in vivo, and have not been created during the process of mechanical isolation, they may play a role in cell-to-cell passage of molecules larger than ions.