ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM⁺/Pax7⁺ (94%), NCAM⁺/Pax7⁻ (4%) and NCAM⁻/Pax7⁺ (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.     

Effects of inhibition of basement membrane collagen deposition on rat mammary gland development

DNA biosynthesis by a system containing giant nuclei isolated from rat trophoblast cells at Day 13 of pregnancy has been studied. A method for the isolation of giant nuclei in good yield has been described. These nuclei were capable of incorporating [³H]dTTP into DNA for 2 hr and the incorporation was proportional to the amount of DNA template (nuclei). The system was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg²⁺ and was stimulated by monovalent ions such as K⁺. The optimum pH was 8.6. The product of the reaction was insensitive to RNase, sensitive to DNase, and banded at 1.710 g/ml in neutral CsCl together with bulk rat trophoblast DNA. Pulse-chase and density labeling experiments utilizing bromodeoxyuridine have indicated that replicative, discontinuous synthesis was taking place at sites previously active in vivo. DNA polymerases α, β, and γ were shown to be present in the nuclei. Experiments utilizing selective inhibitors of polymerases have demonstrated that DNA replication by trophoblast nuclei in vitro was insensitive to the specific α-polymerase inhibitor, aphidicolin, but almost completely inhibited by 2′, 3′-dideoxythymidine 5′-triphosphate as well as by N-ethylmaleimide suggesting that DNA replication observed in these trophoblast nuclei in vitro may be carried out by DNA polymerase γ.     

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G₁/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 M). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×10⁵/root and 1.4×10⁵/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.     

压力超负荷性心肌肥厚大鼠心肌细胞核钙转运的改变

通过腹主动脉缩窄(abdominalaorticcoarctation ,AAC)心肌肥厚大鼠模型制备、差速离心提纯心肌细胞核、酶学方法测定Ca2 +-ATPase活性、45Ca2 +同位素法测定核钙摄取和荧光分光光度计测定细胞核内自由钙浓度 ,初步揭示压力超负荷心肌肥厚大鼠心肌细胞核钙转导异常的环节。结果发现 :心肌细胞核上存在具有[Ca2 +]和ATP依赖性的高亲和力Ca2 +-ATPase ,以[Ca2 +]依赖的方式摄取45Ca2 +,并呈先升高后降低趋势。AAC术后4周大鼠心肌显著肥厚 ,伴有明显的血流动力学异常 ,与对照组比较 ,AAC大鼠心肌细胞核Ca2 +-ATPase活性减少51.93 %(p<0.001) ,但核45Ca2 +摄入量(核外[Ca2 +]浓度为800 -1600nmol/L时)和核内[Ca2 +](核外[Ca2 +]浓度为0 -1000nmol/L时)均明显增加(p<0.05) ;正常组离体心肌细胞核Ca2 +摄取受PKA刺激(p<0.05) ,而被PKC抑制剂和CaM抑制剂显著抑制(p<0.05) ,AAC大鼠心肌细胞核Ca2 +摄取仅受CaM抑制剂抑制(p<0.01) ,而PKA和PKC抑制剂对其无明显影响(p>0.05)。结论为心肌肥厚时 ,心肌细胞核Ca2 +转运系统及其磷酸化调节可能发生改变。     

Purification of Xenopus laevis embryo and liver nuclei using metrizamide.

While the nuclei of many diverse types of tissue can be purified by centrifugation through dense sucrose solutions, Xenopus laevis embryo and liver nuclei present special purification problems due to the presence of large numbers of melanosomes in embryo and liver cells. These melanosomes were removed by isopycnic centrifugation in Metrizamide gradients. In Metrizamide, embryo nuclei banded at an average buoyant density of ρ_5°C = 1.288 ± 0.003 g/cm³. Liver nuclei separated into two peaks, one containing nonparenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.274 ± 0.003 g/cm³ and the other peak containing parenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.284 ± 0.001 g/cm³.     

Transfer of mink genes into mouse cells by means of isolated lipid-encapsulated nuclei

Summary A method for gene transfer by means of interphase nuclei encapsulated within lipid membranes was developed. The method was based on passage of interphase nuclei through a layer of organic solvents containing phospholipids. Evidence was obtained indicating that the nuclei become surrounded by a protective phospholipid membrane: measurements of bound labelled or non-labelled phospholipids; decrease in the permeability of lipid-encapsulated nuclei for high molecular compounds; visualization by direct electron microscopy. Lipid-encapsulated nuclei of mink fibroblasts were used for transformation of mutant mouse LMTK^- cells (deficient for thymidine kinase). The frequency of occurrence of HAT-resistant colonies/recipient cell was 1.9x10^-5. Biochemical analysis of 14 independent clones demonstrated that they all contained TK1 of mink origin. Analysis of 15 other biochemical markers located on 12 of the mink chromosomes revealed the activities of mink galactokinase (a syntenic marker) in 5 transformed clones, and that of mink aconitase-1 (the marker of mink chromosome 12) in 1 clone. No cytogenetically visible donor chromosomes were identified in the transformed clones. Nine transformed clones were tested for the stability of the TK⁺ phenotype; of these, the phenotype was expressed stably in 3 and unstably in 6. The method suggested is similar to the gene transfer procedure using total DNA. Its advantage is in ensuring efficient gene transfer and donor DNA integrity.     

Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs

Abstract: DNA ligase activities were measured in neuron-rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (K_m= 46–48 μM) and bivalent cation (Mg²⁺ or Mn²⁺) were required for the maximal activities in cerebral and liver nuclei. β-Mercaptoethanol did not affect the activities, but N-ethylmaleimide and p-chloromercuribenzoate completely inhibited the activities. Deoxyadenosine-5′-triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na⁺ and Mg²⁺ on the enzyme activities was observed. A high concentration (200 mM) of Na⁺ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM-NaCl. Apparent K_m for the substrate [³²P]phosphoryl DNA was determined according to a modification of the Michaelis-Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar K_m values (about 20 μg of [³²P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher K_m value (54 μg of [³²P]phosphoryl DNA/ml). The modified Michaelis-Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′-phosphomonoester converted into a phosphatase-resistant form per min at 37°C) per μg of nuclear DNA] was 11-fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].     

Mass isolated ameba nuclei. I. Isolation procedure and determination of macromolecular composition and RNA polymerase activity

A rapid method for mass isolation of intact nuclei from Amoeba proteus has been developed. By this procedure, which includes a novel way of recovering nuclei during the filtration step, about 40% of the nuclei in the starting suspension of 4 to 5 × 10⁶ cells can be recovered within 70 min. A typical suspension of purified nuclei consists of 93% intact nuclei, 5.6% food-waste pellets, 0.4% membranes, and 1.0% extracellular debris.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

Rapid determination of chain length pattern in poly(ADP-ribose) samples

(³H)poly(ADP-ribose) synthesized from nuclei by incubation with (³H)NAD was released from protein by alkaline treatment and electrophoresed in dodecyl sulfate gels. Individual polymers up to at least 33 units were completely separated according to their chain length. Size distribution was visualized by fluorography of the gels, and quantified by radioactivity determination of sliced gels The method could be applied to crude nuclear extracts. It showed that nuclei of Ehrlich ascites tumor cells produced a poly(ADP-ribose) pattern distinctly different from that of rat liver nuclei.     

Striatum Adenosine A2 Receptors Are Modified During Seizure: Effect of Cyclopentyladenosine Administration

Rat CNS adenosine A_2A receptors were studied after administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) by means of a quantitative autoradiographic method. Specific binding was quantified in striatum only. The highest density was found in caudate-putamen (2.50 fmol/mm²), followed by nuclei accumbens (1.85 fmol/mm²) and the lowest values in the olfactory tubercle (1.26 fmol/mm²). These differerences were statiscally significant. MP administration (150 mg/kg) caused significant increases (12–18%) in caudate-putamen and nuclei accumbens in both stages: seizure and postseizure and no changes in the olfactory tubercle. CPA administration (2 mg/kg) originated a rise of 16% in nuclei accumbens but no change in the other two regions. When CPA was injected 30 minutes before MP, an increase (18 to 45%) in caudate-putamen and nuclei accumbens at seizure and postseizure stages was observed. Saturation results, in striatal membrane fraction, indicate that receptor sites increased their maximal binding capacity (Bmax) while the apparent dissociation constant (Kd) remained unchanged. These results suggest the involvement of the adenosine A_2A receptors in convulsant activity and that CPA administration at the dose selected brings about a rise in neuronal excitability in this area.     

In vivo development of the lethal yellow (Ay/Ay) mouse embryo at 105 hours post coitum

When histologically examined in utero at 105 hours post coitum embryos from A ^y /a × A ^y /a matings contained a mean (± SE) of 122.7 ± 6.5 nuclei per embryo; of these, 30.2 ± 2.4 nuclei were in the inner cell mass (ICM) and 92.5 ± 4.8 nuclei were within trophoblast cells. Embryos from A ^y /a × a/a matings contained a mean of 141.4 ± 10.6 nuclei per embryo of which 41.7 ± 4.5 nuclei were within the ICM and 99.7 ± 6.9 nuclei were trophoblastic. ICM cell numbers were significantly different between genotypes (P < 0.05) suggesting that homozygous A ^y allele expression selectively interferes with ICM versus trophoblast cell differentiation during preimplantation development.     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000     

Localization of sulfated glycosaminoglycans within cell nuclei by high-resolution autoradiography

Skin fibroblasts cultured from patients with Sanfilippo's disease type B and Hurler's disease, preincubated with [³⁵S]sulfate, were subjected to high resolution autoradiography. A 4-day pulse followed by a 24 h chase resulted in a labelling of 61% of the cell nuclei by silver grains, thus indicating the existence of ethanol-insoluble [³⁵S]sulfate-containing material within the nuclei, especially within their chromatin-rich peripheral zone. A computer-calculated statistical evaluation of the autoradiographic results showed that the silver grains overlying the cell nuclei originated from [³⁵S]radioactivity within the nuclei and not from an overall background or cross fire effects of cytoplasmic radiation sources. Chemical analyses of chloroform/methanol extracts and pronase digests of the [³⁵S]labelled cells provided evidence that neither [³⁵S]sulfatides nor [³⁵S]glycopeptides contribute substantially to the cellular [³⁵S]radioactivity. The results strongly suggest the association of sulfated glycosaminoglycans with cell nuclei.     

Hetero- and euchromatin of synchronous Euglena cells : I. Physical fractionation of nuclei into differentially condensed chromatin

Several attempts were made to isolate intact nuclei and fractionate condensed and extended chromatin from synchronized cells of Euglena gracilis Z. Different factors affecting the recovery and the intactness of nuclei have been tested: detergent concentration, incubation time and addition of Mn²⁺ (0.13 mM) and/or spermidine (0.14 mM) as protective agents. Interphase and mitotic nuclei show preserved chromatin when Mn²⁺ is included, while the combination of Mn²⁺ and spermidine—and, to a lesser extent, spermidine alone—leads to mitotic nuclei with enhanced clumped chromatin. The common procedure to fractionate Euglena chromatin involves swelling of nuclei before disruption. We proved that this step induces artefactual decondensation of packed heterochromatin. Two alternative methods are compared with separate condensed and dispersed chromatin: (1) breakage of swollen nuclei and subsequent addition of divalent cations and/or spermidine with mild pressure shearing forces; (2) disruption of nuclei in a medium containing Mn²⁺ as a protective agent, without swelling. Electron microscopy study indicates that the normal packed appearance of condensed chromatin is preserved, according to the second procedure, while extensive shearing is necessary. Template capacity of the extended chromatin is significantly higher in both methods. Relative amounts of condensed and dispersed chromatin during interphase and mitosis are discussed.     

Nuclear changes induced by the nematodeXiphinema diversicaudatum in root-tips of strawberry

Summary Feeding by the nematodeX. diversicaudatum caused a progressive increase in the DNA content and size of strawberry nuclei. After four days feeding, nuclei had DNA values intermediate between 8C and 16C and had increased in size from a mean of 17 m² for control root tips to 49 m². Multinucleate cells were present after two and four days feeding. There were no ultrastructural differences in the composition of nuclei from control and parasitized root tips, but strawberry nuclei consisted mainly of dispersed chromatin whereas ryegrass nuclei contained a large proportion of condensed chromatin.     

Arginine-rich histones do not exchange between human and mouse chromosomes in hybrid cells

Following division of HeLa-3T3 heterokaryons, human and mouse chromosomes occupy distinct regions within the resulting hybrid nuclei. This favorable orientation of genomes has allowed us to determine whether histones exchange between chromosomes in vivo. Acrylamide gel electrophoresis of the proteins from HeLa cells labeled with ³H-arginine during S phase showed that the core histones were labeled preferentially, constituting 30% of the total cellular tritium and 50% of the label in a crude nuclear fraction. Autoradiographic analysis of cells formed by fusion of ³H-arginine-labeled HeLa cells and 3T3-4E cells showed that ³H-arginine-labeled proteins did not migrate between nuclei in heterokaryons; hybrid cells formed from such heterokaryons contained nuclei in which ³H proteins occupied a sector within the nucleus; “sectored nuclei” could persist for at least 4 days; and the unequal distribution of ³H proteins did not change during DNA synthesis. Electron microscopic examination of hybrid nuclei failed to reveal a physical partition between human and mouse chromosome sets. Sectored nuclei were also observed in synkaryons derived from ³H-arginine-labeled HeLa and unlabeled HeLa cells, indicating that the unequal distribution of ³H-arginine-labeled proteins in HeLa-3T3 hybrid cells did not result from species-specific binding of proteins and DNA. The persistent unequal distribution of ³H-arginine-labeled proteins within hybrid nuclei in the apparent absence of a barrier between mouse and human chromosomes indicates that histones, the principal ³H-arginine-labeled proteins, do not dissociate from DNA in vivo.     

Effect of nuclear Ca2+ uptake inhibitors on Ca2+-activated DNA fragmentation in rat liver nuclei

The effect of nuclear Ca²⁺ uptake inhibitors on the Ca²⁺-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca²⁺ (40 M) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca²⁺-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca²⁺ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 M arachidonic acid, 2.0 mM NAD⁺, 10 M zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca²⁺-ATPase activity and Ca²⁺ uptake in the nuclei, produced a significant inhibition of the Ca²⁺-activated DNA fragmentation in the nuclei. The results show that the Ca²⁺-activated DNA fragmentation is involved in the uptake of Ca²⁺ by the nuclei, suggesting a role of Ca²⁺ transport system in the regulation of liver nuclear functions.