ACTION OF MITOCHONDRIAL DNAASE I IN DESTROYING THE CAPACITY OF ISOLATED CELL NUCLEI TO FORM GELS

1. DNA prepared from non-gelable rat liver nuclei isolated in the presence of disrupted mitochondria at pH 6.0, has been compared with DNA obtained from gelable nuclei isolated at pH 4.0. The DNA of the non-gelable nuclei is partially depolymerized relative to the DNA of the gelable nuclei. 2. It has been found that sufficiently small quantities of crystallized DNAase I can cleave a very large part of the DNA of gelable nuclei isolated at pH 4 from the residual protein of these nuclei without causing extensive depolymerization of the DNA. At the same time the gelable nuclei are rendered non-gelable. 3. Partially purified DNAase II can also render gelable nuclei isolated at pH 4 non-gelable, and in so doing presumably also cleaves the DNA from the residual protein of the nuclei. 4. Mitochondrial DNAase I appears to be the enzyme responsible to a large extent for the cleavage of DNA from the residual protein of gelable rat liver cell nuclei with concomitant destruction of the gel-forming capability of these nuclei, when the nuclei are subjected to the action of disrupted mitochondria at pH 6.0 during the isolation procedure. 5. Mitochondrial DNAase II does not appear to exert appreciable action on nuclei during the course of isolation of the nuclei at pH 6.0 in the presence of disrupted mitochondria. 6. It is probable that DNAase I is not the sole enzyme responsible for destroying the gelability of nuclei isolated at pH 6.0 in the presence of disrupted mitochondria. Protease may be involved. 7. Sodium dodecyl sulfate at pH 6.0–6.3 cleaves the DNA of isolated gelable nuclei from the residual protein of these nuclei over a period of 2 to 3 hours. At pH 7.0–7.5, however, there is negligible cleavage over a period of 96 hours. 8. If non-gelable nuclei are isolated at pH 6.0 in the presence of disrupted mitochondria, DNA subsequently can be removed from them by the use of detergent at pH values ranging from 6.0–7.5 without the necessity of incubation in the detergent solution, since the DNA had already been detached from the residual protein by the action of the mitochondrial enzyme system during isolation of the nuclei.     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇(PEG)诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇（PEG）诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

PROPERTIES OF CELL NUCLEI ISOLATED FROM VARIOUS REGIONS OF RAT BRAIN: DIVERGENT CHARACTERISTICS OF CEREBELLAR CELL NUCLEI

Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus-preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 10⁸ per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non-histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of 'large' and 'small' cell nuclei to cell types in the brain is discussed.     

SOME FACTORS INFLUENCING THE SENSITIVITY OF BODY TEMPERATURE TO ACTIVITY IN NEONATES

In adult humans, core temperature is influenced by activity; the sensitivity of core temperature to such effects shows a phase dependence and is also influenced by the environment and whether the individual is asleep or awake. We have investigated if similar effects are evident in neonates, in whom thermoregulation and the circadian rhythm of core temperature are not fully developed. Eleven full-term, healthy babies were studied singly (light 07:00–19:00) at 2 days of age and again 4 weeks after birth; between these times, they were tended routinely on a communal ward. On study days, 10-minute recordings were made of rectal and skin (abdominal) temperature, heart rate (HR), and behavioral state. Sensitivities of the temperatures to activity (“arousal”) were assessed throughout the 24h by measuring the gradient of (temperature/HR). Sensitivities measured at 01:00, 05:00, 09:00, 13:00,17:00, and 21:00 were used as dependent variables in stepwise regression and linear regression analyses, with “subjects” “light versus dark”, “behavioral state”, and “difference between time of measurement and the acrophase of the endogenous component of the temperature rhythm” (ignoring sign) as possible predictors. (Acrophases of the temperature rhythms had been estimated from 24h data purified using the behavioral state record.) Light versus dark acted as a significant predictor of the sensitivity of rectal temperature to arousal on day 2 and week 4, the sensitivity increasing in the light, and there was limited evidence for behavioral state acting as a predictor on day 2. Neither factor was a significant predictor when the sensitivity of the babies' skin temperatures to arousal was investigated. There was also some evidence that the difference between the time of measurement and the temperature acrophase acted as a predictor of sensitivity to arousal in both rectal (day 2) and skin (week 4) temperature, with larger differences decreasing the sensitivity. These results indicate that there are masking effects on body temperature due to arousal in neonates, the size of which depends on both internal and external factors. However, this sensitivity of temperature to arousal shows differences from the sensitivity of temperature to physical activity in both adult humans and adult mice. One possible explanation of this result is that temperature regulation and the circadian system are not fully developed in humans at this age. (Chronobiology International, 17(5), 679–692, 2000)     

EFFECT OF EHRLICH ASCITES CELL CHALONE ON NASCENT DNA SYNTHESIS IN ISOLATED NUCLEI

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase α and β (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal conditions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [³H]thymidine triphosphate ([³H]-dTTP) incorporation was noted over a chalone range of 50–200 μg/ml. It appears that chalone can inhibit DNA polymerase α directly within the nucleus without an intermediate step such as a cell membrane receptor.     

大鼠大脑皮层细胞核体外转录模型

本文用改良的 Giuffrida法分离纯化大鼠大脑皮层细胞核,产率为41—52％,具有很高的体外转录活性。参照Blatti硫酸铵浓度法建立了大鼠大脑皮层细胞核体外转录模型,能分别测定真核基因三类 RNA聚合酶转录活性,并对有关问题进行了讨论,指出本系统具有操作简便、省时、省实验材料等优点,适用于大脑皮层细胞核体外转录调控的研究。     

心肌细胞核Ca2+库特点及其调节的离体研究

研究核外Ca~(2+)浓度对核Ca~(2+)的影响,及细胞核Ca~(2+)摄取和释放的关系,以探讨核Ca~(2+)转运的调节机制。采用差速离心和密度梯度离心法分离纯化心肌细胞核,以Fluo-4/AM荧光指示剂负载心肌细胞核,应用激光共聚焦扫描显微镜和荧光分光光度计进行观察和测定。结果显示,分离纯化的成年大鼠心肌细胞核内自由[Ca~(2+)]随着核外[Ca~(2+)]的增加而逐渐增加,孵育液[Ca~(2+)]为1000 nmol/L达高峰,但二者增加的程度并不一致,之后随核外[Ca~(2+)]浓度的增加而呈降低趋势。ATP和100—600nmol/L的核外游离Ca~(2+),使心肌细胞核显示核被膜腔Ca~(2+)荧光,ATP和1000nmol/L的核外游离Ca~(2+)则进一步引起核浆内的Ca~(2+)荧光强度升高。荧光染色观察可见IP_3受体染色主要位于核内膜,而钙泵和ryanodine受体染色主要位于核外膜。IP_3和Ryancodine使核Ca~(2+)短暂升高1.68倍和1.93倍(P<0.001),而钙泵抑制剂Thapsigargin和IP_3受体抑制剂Heparin则分别使核Ca~(2+)降低64%和35.6%(p<0.05)。ryanodine使IP_3升高的核Ca~(2+)显著回落至正常水平以下(p<0.001)。Thapsigargin不能阻断IP_3和Ryanodine所致的核Ca~(2+)释放增加(p<0.05),但事先采用钙泵抑制剂Thapsigargin预处理心肌细胞核,则能显著的阻断IP_3和Ryanodine所致的核Ca~(2+)升高作用(Ca~(2+)释放作用)(p<0.05)。结果提示大鼠心肌细胞核可能也是细胞内的钙库之一,心肌细胞核上存在Ca~(2+)-ATPase、ryanodine受体和IP_3受体等Ca~(2+)转运系统,可能参与核Ca~(2+)摄取和释放的调节。     

THE FORMATION OF CDP-DIGLYCERIDE BY ISOLATED NEURONAL NUCLEI

—A population of neuronal nuclei isolated from the rabbit cerebral cortex actively incorporates cytidine-5′-triphosphate into an acid-insoluble product, the incorporation is stimulated by magnesium or manganese ions and appears to utilize CTP directly as substrate. Other nucleoside triphosphates stimulate CTP incorporation at low substrate concentrations, apparently by preventing CTP breakdown, while higher concentrations of nucleoside triphosphates strongly inhibit CTP incorporation at either low or saturating substrate concentrations. CTP incorporation appears to be unrelated to RNA synthesis in that it exhibits a different pH and divalent cation optimum, is unaffected by inhibitors of RNA synthesis, and is strongly inhibited by low concentrations of detergent but not by preincubation of nuclei at 37°C. The product of CTP incorporation is almost entirely soluble in acidified lipid solvents and is not sensitive to digestion by RNAase under conditions where the enzyme can digest newly synthesized RNA. CTP incorporation is markedly stimulated by phosphatidic acid, the stimulated incorporation showing the same characteristics as the unstimulated incorporation, and is inhibited by inositol. Alkaline hydrolysis of the product releases the great majority of radioactivity as 5′-CMP as judged by chromatography and by 5′-nucleotidase action. The product of CTP incorporation migrates with synthetic CDP-diglyceride in five solvent systems. It is concluded that under optimal conditions for CTP incorporation, less than 5% of the incorporated CTP can be included in a polynucleotide chain, this small proportion of the total incorporation could either represent residual RNA synthesis or the formation of poly (C) with an average chain length of less than 3 residues. Under optimal conditions the great majority of CTP incorporation by neuronal nuclei in the presence of either magnesium or manganese ions represents the formation of the unique liponucleotide CDP-diglyceride.     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

THE DIFFERENTIATING ABILITY OF RAT LENS EPITHELIAL CELLS IN CELL CULTURE

Dissociated cells of lens epithelia of adult rats were monolayerly cultured in vitro. After about 15–20 days' period of active cell growth, such characteristic structures that correspond to "lentoid bodies" described previously in chick cultures were formed. These structures consisted of elongated cells, ultrastructural profile of which was similar with lens fiber. The presence of gamma-crystallin, a marker molecule specific to mature lens fiber, was confirmed in these elongated cells by means of fluorescent antibody technique. The differentiation of lens fiber in vitro was also recognized in clones originating from single lens epithelial cells cultured at very low cell density.     

乌梁素海野生芦苇群落生物量及影响因子分析

 对内蒙古乌梁素海湿地野生芦苇(Phragmites australis)生物量的调查基础上,探讨了富营养化湖泊湿地水体的物理化学性质对芦苇生物量的影响。结果表明：1）由于环境因子的影响,芦苇群落生物量变化较大,介于1.73～3.00 kg·m-2之间;地下和地上生物量之比介于1.14～2.19之间;2）芦苇群落生物量受多种因素的影响,其中水深是最主要的限制因子,水上生物量和地上生物量随着水深的增加而增加,而地下与地上生物量的比值则随水深的增加而减少,这主要是由于水深改变了芦苇群落的结构（群落密度）和个体形态(株高和株茎);3）芦苇群落生物量随着水体N浓度增加而增加。芦苇各器官（叶、茎、根状茎和根）的N∶P为7.59～12.21,小于14,这也说明该水体中的N负荷是影响芦苇生长的主要限制因子;4）土壤有机质分解对芦苇生长没有产生毒害作用。     

STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study

Neurons of the mouse were labeled with [³H]thymidine during their prenatal period of proliferation. The ³H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the ³H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [³H]β self-absorption in the material of the sections. Injection of a high dose of [³H]thymidine into young adult mice did not result in ³H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.