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1.
The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl2 and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:
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2.
Autophagic vacuoles (AV) were purified from livers of rats which were pretreated with vinblastine (VBL) to increase the occurrence of AV. To measure proteolysis in the isolated AV rats were labelled with [14C]leucine 2 or 16 h before sacrifice. The integrity of the AV was studied by measuring the leakage of hydrolytic enzymes during incubation at various pHs. VBL causes an increase in the degradation rate of liver homogenate and isolated AV. This increase was moderate if proteolysis was measured at neutral pH, whereas adjustment to acidic pH enhanced the rate of autodegradation in the AV several-fold. This indicates that the VBL-induced AV have acquired hydrolytic enzymes either by fusion with lysosomes or possibly by the sequestering endoplasmic reticulum (ER) membranes forming the limiting membranes of the AV. The internal pH is not optimal for degradation in vitro of sequestered proteins, indicating insufficient acidification of the isolated AV. Lysosomotropic inhibitors, like chloroquine and propylamine, but not asparagine, impede proteolysis in isolated AV, but not more than 40%.  相似文献   

3.
Treatment of Xenopus laevis follicles with 50–100 units/ml of human chorionic gonadotropin causes rapid stimulation of [14C]glucose uptake. Studies with these follicles showed that the stimulation of uptake occurred with a wide range of concentrations of [14C]glucose or its nonmetabolizable analog [14C]3-O-methylglucose. Approx. 70% of the glucose taken up in both hormone-treated and control cells becomes incorporated into glycogen within 1 h. The uptake of sugar by these follicles was also stimulated by bovine-luteinizing hormone—but not by folliclestimulating hormone, progesterone or insulin. Human chorionic gonadotropin stimulated sugar uptake by follicles containing medium-sized oocytes (stages 3,4 and 5 according to Dumont) which cannot be induced to undergo meiotic maturation by this hormone. After 4–6 h treatment of fully grown X. laevis follicles with either progesterone or human chorionic gonadotropin, glucose uptake suffers a drastic decrease to below basal levels. This inhibition of uptake is coincident with the breakdown of the germinal vesicle of the oocyte and is clearly related to meiotic maturation, since it is not observed with medium-sized follicles which cannot mature.  相似文献   

4.
Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.  相似文献   

5.
A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.  相似文献   

6.
Isolated nuclei of plant (Daucus carota) protoplasts were injected into immature and maturing Xenopus oocytes. In the first series the transplanted nuclei continued to synthesize RNA for at least 18 h, whereas in the latter series the nuclear membrane was disrupted and the chromatin was induced to condense prematurely within 30 min. The results of this study suggest that fundamental biological processes like chromosome condensation during mitosis and meiosis —which are no different in animal and plant kingdom from a morphological point of view—are also very similar with respect to the underlying regulatory control.  相似文献   

7.
Compact mouse morulae were decompacted in calcium-free medium and allowed to recompact in standard embryo culture medium. When the recompaction medium contained trifluoperazine (TFP)(0.05 mM), an inhibitor of the calcium-dependent protein calmodulin, the embryos failed to recompact. This effect could not be overcome by either db-cAMP (1.0 mM) or theophylline (0.75 mg/ml). When the recompaction medium contained less than standard calcium (0.085 or 0.17 mM) the embryos recompacted at a slower rate than in control medium (1.7 mM). The calcium ionophore A23187, at concentrations up to 1.5 X 10(-3) mM, had no significant stimulatory effect upon the recompaction rte of embryos in the reduced calcium medium. In addition, the calcium antagonist Verapamil (0.3 mM), which blocks calcium uptake by cells, significantly inhibited recompaction in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. We conclude that calcium uptake into the cytoplasm is required for recompaction, but that cell surface calcium is also required and is rate-limiting under these experimental conditions.  相似文献   

8.
A cloned population of mouse C3H/He keratinocytes was obtained from the 14th passage of an epidermal cell line. A two-step cloning procedure using Petriperm dishes was performed. The cloned population, grown at 34 °C, was subcultured more than 30 times over a one year period. By day 14, three cell layers were formed; the ultrastructural morphology and immunofluorescence characterization of these layers showed numerous tonofilament bundles and well organized desmosome tonofilament structures. They thereby resemble the proliferative compartment of the epidermis. High resolution acrylamide gel electrophoresis of the keratins extracted from the cloned cells showed the presence of many keratin subunits. The tonofilaments extracted from the cell layers, as well as from the supernatant cells, contained a small quantity of high MW keratins (rel. MW 63 000; apparent isoelectric point 5.5–6.2). These results indicate that the cloned keratinocyte cell line had retained a certain maturation capacity in culture.  相似文献   

9.
Human monocyte-derived macrophages were demonstrated to have separate and morphologically distinct binding sites for low density lipoprotein (LDL) and acetylated LDL (AcLDL). Using an indirect immunoperoxidase technique and electron microscopy, only LDL was shown to bind to its receptor in coated pits on the macrophage membrane, whereas the distribution of AcLDL-receptor complexes was dependent upon whether or not the cells were fixed prior to incubation with AcLDL. In cells incubated with AcLDL, then fixed, electron-dense precipitate was found in aggregates, sometimes near pseudopodia; fixed cells incubated with AcLDL had electron-dense precipitate more uniformly spread along the membrane. These data suggest that the 'scavenger' receptor is diffusely distributed in the membrane and that following AcLDL binding the receptors cluster in regions of the membrane which do not contain coated pits.  相似文献   

10.
An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.  相似文献   

11.
DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(106) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.  相似文献   

12.
In a recent communication it was shown that intravenously injected radioactively labelled hyaluronic acid was preferentially taken up by the liver and degraded. We now report that uptake occurs in the liver endothelial cells and that these cells degrade the polysaccharide in vitro into low-molecular weight (LMW) products.  相似文献   

13.
DNA synthesis was studied during the overmaturational stage of starfish eggs. Some DNA synthesis took place in the overmature eggs of the starfish. The DNA synthesis was resistant to aphidicolin. Judging from its circularity and small molecular size, the DNA synthesized is of mitochondrial origin.  相似文献   

14.
15.
Rabbit antiserum against rat plasma fibronectin induced histamine release in isolated rat peritoneal mast cells. Immunofluorescence revealed fibronectin on the mast cells in rat mesentery and on the surface of the isolated mast cells. Mast cells adhered to collagen-coated dishes. This cellular adherence was inhibited by the addition of anti-fibronectin. Fibronectin on the surface of mast cells may play a role of attachment of the cells to collagenous connective tissues.  相似文献   

16.
Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca2+-free sea water (a treatment that bypasses the cortical reaction and the Ca2+ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.  相似文献   

17.
A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of 35SO4?2, [3H]-glucosamine and [3H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.  相似文献   

18.
Cloned variants of a rat hepatoma cell line have been isolated which exhibit normal attachment and spreading behavior on fibronectin substrata, but which are defective in their ability to attach to native collagen films. These clones should be useful for identifying specific macromolecules involved in the cell-to-collagen interaction.  相似文献   

19.
Tetrahymena calmodulins from cilia, cell bodies and whole cells were isolated separately and compared. These calmodulins showed just the same properties: they co-migrated in SDS-polyacrylamide gel electrophoresis, had a Ca2+-dependent electrophoretic mobility change in alkali gel, held the same antigenic determinants in common, and activated brain cyclic nucleotide phosphodiesterase Ca2+-dependently with identical activation curves. Distributions of calmodulin and calmodulin-counterpart in Tetrahymena cilium were investigated by using alkali gel electrophoresis in the presence of Ca2+ or EGTA, and by immunoelectron microscopy. Calmodulin was detected in the membrane plus matrix fraction and outer-doublet microtubule fraction, and its Ca2+-dependent counterpart existed exclusively in the latter fraction. However, neither calmodulin nor its counterpart was detected in the crude dynein fraction. Immunoelectron microscopy revealed that calmodulin was localized along the longitudinal axis of outer-doublet microtubules at regular intervals of about 90 nm. The calmodulin-binding site in the ciliary axoneme was suggested to be interdoublet links.  相似文献   

20.
Pre-leptotene primary spermatocyte %Pachytene primary spermatocyte %Round spermatid %Elongated spermatid %
DNA polymerase α2542303
DNA polymerase β2934361
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