d-ASPARTATE OXIDASE ACTIVITY IN EXTRACTS OF MAMMALIAN CENTRAL NERVOUS TISSUE

Abstract— d -Aspartate oxidase activity has been measured in water extracts of acetone powders prepared from cat forebrain, cerebellum and spinal cord, rat brain, hog brain and sheep brain stem, and compared with that found in rabbit and cat kidney. The results suggest that the brain enzyme has very similar properties to the n-aspartate oxidase ( d -aspartate: oxygen oxidorcductase (deaminating), EC 1.4.3.1) of kidney. Crude extracts (ammonium sulphate fractions of water extracts of acetone powders) displayed little activity without added FAD. FMN could not replace FAD. With oxygen as electron acceptor, the enzyme oxidized d -aspartate much more rapidly than d -glutamate, and displayed quite high activities with N -substituted derivatives of d -aspartate as substrates. Those amino acids susceptible to oxidation by d -amino acid oxidase were not oxidized by the d -aspartate oxidase. The regional distribution of the d -aspartate oxidase activity within the CNS differed from that of d -amino acid oxidase. As has been previously observed for kidney d -aspartate oxidase activity, dicarboxylic acids competitively inhibited this enzymic activity in brain extracts, while sodium benzoate and sodium barbitone, inhibitors of d -amino acid oxidase, were without effect.     

CTP SYNTHETASE ACTIVITY IN NEONATAL AND ADULT RAT BRAIN

—The activity of CTP synthetase (UTP:ammonia ligase (ADP-forming), EC 6.3.4.2) in adult and new-born rat brain was determined by an enzyme assay using [¹⁴C]UTP as a substrate. The activity was age-dependent and showed a distinctive distribution pattern between the cerebral hemispheres and cerebellum. The possible correlations between the activity of CTP synthetase and the rate of RNA or lipid biosynthesis, as well as the regulatory importance of the enzyme in the formation of cytidine nucleotides are discussed.     

LEVEL AND AMINO ACID ACCEPTOR ACTIVITY OF MOUSE BRAIN tRNA DURING NEURAL DEVELOPMENT

Abstract— The level of tRNA in mouse brain tissue was measured at various stages of postnatal development. The amount of tRNA per unit of brain wet weight was little, if at all, altered during the first 22 days after birth and decreased by 26 and 32 per cent by 56 days and maturity, respectively. On a DNA or cellular basis, there was no maturation-dependent decrease in tRNA content. The total amino acid acceptor activity of tRNA for seven different amino acids was measured during neural development. There were considerable differences in the tRNA acceptor activities of individual amino acids within an age group; however on a DNA basis, there was little difference between tRNA preparations obtained from newborn and adult mouse brain tissue. The in vivo levels of aminoacylated-tRNA for the seven amino acids of interest, were measured in brain tissue of 1–, 9–, 34, 70–day-old and adult (over 9 months old) mice. Alterations in tRNA level, total tRNA acceptor activity, for each amino acid, and the levels of in uivo aminoacylation of tRNA were shown to be independent of developmental alterations in brain amino acid pool sizes. The results are discussed with regard to the availability of cellular amino acids for translational events during early mammalian brain development.     

ARCHITECTURE AND NERVE SUPPLY OF MAMMALIAN SMOOTH MUSCLE TISSUE

Smooth muscle tissue from mouse urinary bladder, uterus, and gall bladder has been studied by means of the electron microscope. The smooth muscle cells are distinctly and completely separated from each other by a cytolemma comparable to the sarcolemma of striated muscle. The tissue is thus cellular and not syncytial. With this evidence, supported by electron microscopy of other tissues, we question the existence of true syncytia in animal tissues. Individual cell membranes necessary for the electrophysiologic events exist in smooth muscle, and its nerve and conduction in a tissue such as uterus or bladder can occur at the cellular level as well as at the tissue area level. The smooth muscle cell contains myofilaments, nucleus, endoplasmic reticulum, mitochondria, Golgi complex, centrosome, and pinocytotic vesicles. These structures are described in some detail, and their probable interrelations and functions are discussed. The autonomic nerves innervating smooth muscle cells are composed of axons and lemnoblasts. The axon is suspended by the mesaxon formed by the infolded plasma membrane of the lemnoblast. The respective plasma membranes separate axon and lemnoblast from each other and from surrounding muscle cells. The axons of autonomic nerves never penetrate the plasma membrane of the muscle cell, but pass or intrude into muscle cell pockets, forming a contact between axonal plasma membrane and smooth muscle plasma membrane. The lemnoblast shows well developed endoplasmic reticulum with Palade granules, mitochondria, and a long, elliptical nucleus. The axon contains neurofilaments, mitochondria, and synaptic vesicles; the quantity of the latter two being significantly greater in the periphery of lemnoblasts and near axon-muscle contact regions. We regard the contact regions as the synapses between the autonomic nerves and the smooth muscle cells.     

FLAME PHOTOMETRIC ANALYSIS OF SODIUM AND POTASSIUM IN NANOGRAM SAMPLES OF MAMMALIAN NERVOUS TISSUE

A dual-channel integrating micro-flame-photometer was evaluated for simultaneous analysis of sodium and potassium in aqueous extracts from nanogram samples of frozen-dried mammalian nervous tissue. Calibrated quartz constriction micropipettes delivered 10⁻⁸ l. of extraction fluid to a 100 μ platinum-iridium wire for insertion directly into the flame. Over-all reproducibility was 4 per cent for twenty samples containing 6.5 × 10⁻¹¹ g K and 2.2 × 10⁻¹¹ g Na. Large amounts of anions decreased the emissions for both sodium and potassium, but no interference between sodium and potassium was found over the range adopted for biological analyses. The micro-flame-photometer gave results for a few nanolitres of aqueous extracts of brain homogenates which were within 3-5 per cent of those obtained on larger volumes with a conventional flame photometer. Macroanalysis and microanalyses of microgram quantities of frozen-dried tissue sections of cerebral cortex were also in agreement. Nanogram samples from frozen-dried spinal ganglia of a rabbit gave average values for sodium and potassium (calculated/g wet wt.) which were similar to those for aqueous extracts of rabbit brain homogenates. Samples from peripheral ganglia in vivo, 10 minpost mortem and 20 min post mortem had significantly different average K/Na ratios of 1.97, 2.64 and 3.23, respectively.     

STUDIES ON THE INTRACELLULAR DIGESTIVE PROCESS IN MAMMALIAN TISSUE CULTURE CELLS

DNA-protein coacervates containing colloidal gold particles were readily phagocytized by strain L fibroblasts. During the subsequent digestion process, the gold particles served as markers which permitted the demonstration of the evolution of digestive vacuoles to multivesicular bodies and finally to dense bodies. Acid phosphatase and esterolytic activity was present in these structures. The hydrolytic enzymes were apparently brought to the phagocytotic vacuoles in small vesicles originating in the Golgi region. These vesicles entered the vacuoles prior to the digestion of the coacervates and the appearance of positive cytochemical reactions. The cytoplasmic dense bodies frequently merged with the phagocytotic vacuoles. This was demonstrated by prelabeling the dense bodies with colloidal iron prior to phagocytosis of the coacervates. In addition, evidence is presented for the interrelationship of the phagocytotic and autophagic pathways.     

新生大鼠窦房结组织、培养细胞的glycogen,LDH和SDH定量形态学研究

本实验采用窦房结细胞纯化培养、ＰＡＳ、Ｐｅａｒｓｏｎ和Ｐｒｅｓｔｏｎ反应以及图像分析等方法,研究了新生ＳＤ大鼠窦房结细胞的糖原（ｇｌｙｃｏｇｅｎ）、琥珀酸脱氢酶（ＳＤＨ）,和乳酸脱氢酶（ＬＤＨ）的分布和含量。结果显示,在窦房结组织中的糖原（／）与心房中（／）、心室中（）含量近似;ＳＤＨ含量和活性（＋／）与心房中（＋／）几乎相等,但明显低于心室中（／）;ＬＤＨ的含量和活性（／）比心房中（／＋）的稍高,但明显高于心室中（＋／－）。图像分析结果是：培养窦房结细胞ＳＤＨ含量和活性显著低于心室肌细胞中的ＳＤＨ（Ｐ＜０．０１）,而ＬＤＨ含量和活性显著高于心室肌细胞中的ＬＤＨ（Ｐ＜０．０１）。本文还对ＳＤＨ、ＬＤＨ在窦房结的分布及生理作用进行了讨论。     

ACETYL- AND BUTYRYLCHOLINESTERASE ACTIVITY OF SELECTED BRAIN AREAS IN DEVELOPING RATS AFTER NEONATAL X-IRRADIATION*

Acetylcholinesterase and butyrylcholinesterase activities in sensori-motor cortex, hypothalamus, cerebellum, and brain stem were compared in normally developing Long-Evans rats and after neonatal whole-body exposure to 450 r X-radiation. Enzyme activities were measured on three postnatal days: day 10, when brain is still immature; day 24, when it has reached functional and morphological maturity; and day 64, after sexual maturation. In controls, acetylcholinesterase and butyrycholinesterase activities increased with age in all areas, especially between 10 and 24 days; e.g., in sensori-motor cortex acetylcholinesterase activity increased 60 per cent from 10 to 24 days and 12 per cent from 24 to 64 days. At all ages acetylcholinesterase activity was highest in the brain stern, followed in decreasing order by the hypothalamus, cerebellum, and sensori-motor cortex. Butyrylcholinesterase activity was higher in subcortical than in cortical areas. In neonatally irradiated rats, acetylcholinesterase activity was significantly decreased in the ontogenetically newer structures at 10, but not at 64, days; in the hypothalamus, it remained normal at 10 days but was significantly decreased at 24 and 64 days. Butyrylcholinesterase activity was significantly decreased in some areas 1 week after radiation but returned to normal at 24 days. Total esterase activity in whole blood was signtficantly decreased at 10 days in irradiated rats but returned to control levels by the end of the experiment. The greatest post-radiation decline in acetylcholinesterase activity (60 per cent below controls) did not result in spontaneous gross behaviour alterations, but may be related to disturbances in functional brain maturation evidenced by specific tests. If the role of acetycholine as a central neurotransmitter is accepted, these data suggest that radiation alters acetycholine/acetylcholinesterase ratios and thereby cholinergie synaptic transmission.     

新生大鼠窦房结组织和培养细胞的心钠素(ANF)表达的定量形态学观察

本实验采用窦房结细胞纯化培养、免疫细胞化学、免疫组化、免疫电镜 （ｐＡｇ 技术） 和图像分析等多种方法,研究了新生ＳＤ大鼠窦房结的ＡＮＦ表达。结果提示： 窦房结原位组织和培养细胞的胞浆内均可见ＡＮＦ阳性反应颗粒。培养细胞的核附近电子致密颗粒有１０ｎｍ ＡＮＦ免疫反应的ｐＡｇ 颗粒。窦房结ＡＮＦ的含量和活性明显低于心房 （Ｐ＜ ００１）。本文还对新生鼠窦房结ＡＮＦ的生理作用进行了讨论     

LOCALIZATION OF HEXOKINASE IN NEURAL TISSUE: LIGHT MICROSCOPIC STUDIES WITH IMMUNOFLUORESCENCE AND HISTOCHEMICAL PROCEDURES

Abstract— The distribution of hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) in rat cerebellum, retina, hippocampus, choroid plexus and ependymal cells of the cerebral ventricles, and dorsal root ganglion has been determined, at the light microscopic level, by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. With the exception of an artifactual staining of the outer photoreceptor segments of retina when the histochemical procedure was used, both methods gave comparable results, from which the following conclusions are drawn:

• (a) The cytoplasm of neuronal cell bodies clearly contained hexokinase, although the relative levels varied markedly among different types of neurons; such variations have previously been detected by direct assay of hexokinase in dissected neuronal cell bodies (Kato & Lowry , 1973a).
• (b) Glial cells contained readily detectable levels of hexokinase: the immunofluorescence technique revealed spidery glial processes within the myelinated tracts; in other areas, glial cell cytoplasms were indistinguishable from surrounding neuropil, indicating comparable levels of hexokinase; the satellite glia of dorsal root ganglia actually contained higher levels than did adjacent large neurons. The present results, therefore, do not support previous suggestions that glia are characteristically low and neurons characteristically high in hexokinase content.
• (c) Hexokinase was distributed throughout neuropil areas, with a somewhat speckled appearance suggesting the existence of small localizations of relatively higher activity, the nature of which could not be determined at this level of resolution; the hexokinase level in neuropil was clearly higher than that of white fiber tracts, in agreement with previous direct biochemical measurements (Buell et al., 1958)
• (d) No detectable levels of hexokinase were found in cell nuclei.
• (e) Regions expected to be rich in nerve terminals (e.g. the cerebellar glomeruli, the plexiform layers of retina) showed relatively high hexokinase levels compared to the cytoplasm of adjacent neuronal perikarya, in agreement with previous subcellular fractionation experiments which indicated relatively high levels of hexokinase in nerve endings (Wilson , 1972). Considered along with the‘high affinity’glucose transport system in nerve endings (Diamond & Fishman , 1973), these results suggest nerve terminals are well adapted for the'efficient acquisition and introduction of glucose into metabolism.
• (f) In addition, high levels of hexokinase were observed in the inner photoreceptor segments of retina, and in the ependymal and choroid plexus cells of the ventricles.

     

METHOD FOR COMBINED ULTRASTRUCTURAL AND BIOCHEMICAL ANALYSIS OF NEURAL TISSUE

No significant change was found in the electrolytes and lipids of the brain analyzed after glutaraldehyde fixation by perfusion of laboratory animals; such fixation also satisfactorily preserves neural tissues for electron microscopy. The brains of normal and tumor-bearing C3H mice, Wistar rats, and New Zealand rabbits were studied. Little difference was found in the dry weight and the content of sodium, potassium, total lipid and lipid fractions, and in the sulfate space (S³⁵O₄) between specimens from unperfused and perfused animals, whether normal or tumor-bearing. The results suggest the possibility of using selected regions of the nervous system, dissected after fixation, for chemical study and at the same time characterizing similar regions morphologically with the electron microscope.     

EFFECT OF DEXAMETHASONE ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE IN CHROMAFFIN TISSUE OF THE NEONATAL RAT

Abstract— Treatment of neonatal rats with dexamethasone resulted in the appearance of phenylethanolamine- N -methyltransferase (PNMT) and numerous small, intensely fluorescent (SIF) cells in abdominal paraganglia and in sympathetic paravertebral ganglia. These cells may be derived from a primitive stem cell precursor, but because of their unusual anatomical features, origin from ganglion cells cannot be altogether ruled out. Associated with the proliferation of the cells was a marked increase in the PNMT activity of the tissues. The PNMT response to the glucocorticoid was limited to the first few days of life, as was the SIF cell response. After discontinuance of dexamethasone, the enzyme activity fell very rapidly, while the number of cells declined at a slower rate.     

催产素及其受体与哺乳动物的生殖

催产素（OT）是一种9肽激素,主要由哺乳动物下丘脑产生,以神经内分泌,旁分泌或自分泌形成,在哺乳动物生殖过程中发挥重要作用,催产素受体（OTR）是与G－蛋白相耦联的膜蛋白,通过激活磷脂酶C发挥其生理作用,OT在交配,分娩,哺池时由神经垂体（垂体后叶）脉冲式释放,促进子宫平滑肌和乳腺肌上皮细胞收缩,利用精子运行,胎儿娩出和射出乳汁,OT在中枢神经系统中参与调节母性行为,在性腺中促进某些物种的黄体形成,OT与PGF2a共同作用使有蹄动物黄体退化,以上过程都依赖于OT和OTR基因的时空特异性表达,多种激素参与它们的表达调控,但OT的生理作用有时也可被其它途径所替代。     

中国地质事件与哺乳动物的分布

自第三纪初开始,古地中海(Tethys)消亡,欧亚板块与印度板块合并至今,中国大陆的地质构造运动与古地理环境变迁大势,已为许多有充实基础的地质学研究所揭示,且普遍被地学界所接受.这一情况使我们可以在信赖地质-古地理事件研究成果的基础上,讨论生物的分布,而避免在生物地理与古环境之间产生循环论证的危险.本文列举的事实,说明了我国因地质事件而产生的自然环境分化与变迁对动物分布的影响.概括而言,它表现为三种与动物扩散(dispersal)能力相联系的效应:有效的阻障(barrier)效应;部分的阻障效应,与此相联系的是过渡(transition)现象;走廊(corridor)或过滤(filter)效应.喜玛拉雅-秦岭-淮河一线是一条重要的地形-气候分界线,始于上新世,延伸至今.其阻障效应反映在动物区系上古北与东洋的明显分化和相应的分布型的形成.在整个第四纪内,随时间的推移,青藏高原的抬升,喜马拉雅段不断强化其阻障效应,中亚地区的干旱逐渐加强,一个适应干旱条件的动物区系随之形成.而在秦岭段所在的东部季风区发生过数次自然地带的南北推移,结果形成了此两大区系的过渡.正如Darlington(1957:472)所说广泛而充分的过渡(full transition),以致对古北与东洋两大区系在此季风区的划分意见产生较大的分歧.但是,沿秦岭-淮河一线仍有一两大区系的明显消减带,表明其对不同类群的动物仍有不同程度的即部分的阻障作用,可以作为古北与东洋两界在本地区的分野.黄土高原与华北平原半干旱-半湿润环境的形成是对基本上属于湿润环境的季风区的干扰.它一方面是喜湿动物的阻障,导致南北方向扩散(dispersal)的中止或间断(disjunction),另一方面成为干旱成分向东扩散的通道.伴随着青藏高原而形成的横断山系,其走向与喜马拉雅相反.地形上的这一特点,使其形成了南北动物扩散上的过道或阻障上的缺口.此山系的垂直自然地带,为动物在生态上提供了多种栖息环境.横断山系垂直幅度大,且位于中低纬度,在自更新世以来世界性的气候变迁中,山地景观带只引起小幅度的、以百米计的垂直移动,不象北方或开阔景观中大幅度的水平推移,因而最大限度地保存了原始的而且是世质的生存环境.这种多样性的和在历史变迁中相对稳定的环境,对物种的保存和分纶都是十分有利的.陆栖脊椎动物中许多类群的物种密度,在本山系均达到最高的事实,可以此假设提供佐证.总的说来,中国哺乳动物动物的现代分布的9个主要类型,其分布范围大体上分别于不同尺度的自然地理环境相一致,是长期以来适应地质-古地理变迁的结果.     

VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

EFFECTS OF ACUTE HYPERTHERMIA ON POLYRIBOSOMES, IN VIVO PROTEIN SYNTHESIS AND ORNITHINE DECARBOXYLASE ACTIVITY IN THE NEONATAL RAT BRAIN

—Acute hyperthermia produces in situ disaggregation of brain polyribosomes in infant rats, as determined by electron microscopy. Protein synthesis is inhibited in infant, but not weanling, rat brain by 45 min of hyperthermia; this inhibition is reversed during a 2 h recovery period at normothermic conditions. Hepatic protein synthesis was inhibited less than that of brain. Acute hyperthermia also leads to a profound loss of ornithine decarboxylase activity in brain; during recovery the activity of this enzyme overshoots to values greater than those of normothermic control rats. This increase is blocked by cycloheximide administration. In testis, a tissue with high ornithine decarboxylase activity, enzyme activity was not affected by hyperthermia and recovery, indicating tissue specificity for these effects.     

NEURAL BASIS AND BIOLOGICAL FUNCTION OF MASKING BY LIGHT IN MAMMALS: SUPPRESSION OF MELATONIN AND LOCOMOTOR ACTIVITY

Light influences mammalian circadian rhythms in two different ways: (1) It entrains endogenous oscillators (clocks), which regulate physiology and behavior; and (2) it affects directly and often immediately physiology and behavior (these effects are also referred to as masking). Masking effects of light on pineal melatonin, locomotor activity, and the sleep-wake cycle in mammals and man are reviewed. They seem to represent a universal response in this group. The review reveals that the mechanism of photic inhibition of melatonin is fairly well understood, whereas only little is known about the influence of light on other circadian rhythm outputs, such as locomotor activity. (Chronobiology International, 18(5), 737-758, 2001)     

NEURAL BASIS AND BIOLOGICAL FUNCTION OF MASKING BY LIGHT IN MAMMALS: SUPPRESSION OF MELATONIN AND LOCOMOTOR ACTIVITY

Light influences mammalian circadian rhythms in two different ways: (1) It entrains endogenous oscillators (clocks), which regulate physiology and behavior; and (2) it affects directly and often immediately physiology and behavior (these effects are also referred to as masking). Masking effects of light on pineal melatonin, locomotor activity, and the sleep-wake cycle in mammals and man are reviewed. They seem to represent a universal response in this group. The review reveals that the mechanism of photic inhibition of melatonin is fairly well understood, whereas only little is known about the influence of light on other circadian rhythm outputs, such as locomotor activity. (Chronobiology International, 18(5), 737–758, 2001)