Effect of 1-beta-D-arabinofuranosylcytosine triphosphate on DNA synthesis in isolated HeLa cell nuclei

1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (ara-CTP) was found to be a competitive inhibitor of DNA synthesis in nuclei isolated from synchronized S-phase HeLa S3 cells. The inhibition was reversed by dCTP. The main inhibitory effect was to reduce the rate of elongation of the primary DNA pieces from ara-CTP-treated nuclei after a 60-s pulse was about 115-120 nucleotides vs. 215-220 in control nuclei. A reduction in the frequency of initiation of primary DNA pieces probably also occurred. A slight inhibition of the transfer of labeled material from the primary pieces into high-molecular weight DNA was noted and probably arose from the fact that the reduced rate of growth of the primary pieces caused the critical size for ligation to be reached later. Synchronized HeLa S3 cells in suspension were treated with 1-beta-D-arabinofuranosylcytosine for 2 h in S-phase to give a 99% inhibition of DNA synthesis prior to isolation of nuclei. Neither the rate nor the extent of [3H]TTP incorporation into DNA decreased when such nuclei were incubated in the absence of ara-CTP, but the rate of ligation of primary pieces was reduced.     

ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei

We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.     

Viral DNA synthesis in vitro with the inclusions isolated from adenovirus 12-infected cells

A fraction defined as the inclusions was isolated by banding in CsC1 gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18S, 22S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.     

A chromosome 4 satellite I DNA isolated from SV40-transformed human cells

Anticancer inhibitors of topoisomerase I (TOP1, EC 5.99.1.2) cause the reversible stabilization of the TOP1-DNA covalent complex (cleavable complex). The cleavable complex can be converted into a double-strand break, the presumed cytotoxic lesion, by active replication forks. Cytotoxicity independent of DNA replication has also been demonstrated, and suggested to have possible clinical significance. To assess the importance of the replication-independent mechanism of camptothecin (CPT) cytotoxicity we have analyzed replication-dependent and replication-independent cytotoxicity following a brief CPT treatment (40 min) of seven human colon tumor cell lines. The cell lines were exposed to CPT in the presence or absence of aphidicolin, an inhibitor of DNA polymerases alpha, delta or epsilon. The seven cell lines responded similarly to CPT: treatments of less than 0.5 microM caused cytotoxicity only when DNA replication was ongoing, as evidenced by a plateau in the cytotoxicity curve corresponding to the S-phase fraction and the prevention of this cytotoxicity by aphidicolin cotreatment; at higher CPT doses, the cytotoxicity exceeded the S-phase fraction and was not prevented by aphidicolin. The CPT sensitivity among the cell lines, measured as the concentration required to inhibit cell growth by 25%, was between 0.17 and 0.43 microM without aphidicolin and 2-10 microM with aphidicolin cotreatment; with aphidicolin in cotreatment, 20-fold greater CPT concentrations were required, on average among the cell lines, to achieve cytotoxicity equivalent to CPT treatment alone. The potential of the lower dose and longer duration treatments of camptothecins used in the clinical setting to produce cytotoxicity independent of DNA replication is discussed.     

Non-enzymatic peroxidation of lipids isolated from rat liver microsomes, mitochondria and nuclei

AIM: To compare lansoprazole 30 mg daily with ranitidine 150 mg b.d. in the treatment of acid-related dyspepsia in general practice. METHODS: In a double-blind, parallel group, randomized, mutlicentre study conducted in 32 general practices in the UK, 213 patients were randomized to receive lansoprazole 30 mg daily, and 219 to receive ranitidine 150 mg b.d., for 4 weeks. All patients had experienced symptoms of reflux-like or ulcer-like dyspepsia on at least 4 of the 7 days prior to the study; 75% had experienced dyspepsia in the past, and 74 of the lansoprazole patients and 77 of the ranitidine patients had documented histories of acid-related disorders, investigating by either radiology or endoscopy. RESULTS: After 2 weeks 55% of the lansoprazole patients and 33% of the ranitidine group were symptom-free (P = 0.001, chi 2 = 7.12) with corresponding 4-week figures of 69% and 44%, respectively (P = 0.001, chi 2 = 18.03). Similar figures were found at both 2 and 4 weeks for daytime and night-time heartburn and epigastric pain scores; in the lansoprazole group, at 4 weeks, 80% of patients were free of daytime heartburn and 81% of night-time epigastric pain, compared with 55% (P = 0.001, chi 2 = 15.44) and 65% (P = 0.01, chi 2 = 6.10) in the ranitidine group. CONCLUSION: Superior symptom relief for patients presenting with ulcer-like and reflux-like symptoms in general practice is provided by lansoprazole 30 mg daily compared with ranitidine 150 mg twice daily.     

Preparation of high molecular weight genomic DNA from nuclei of woody plants

We have developed a rapid and reliable method for preparation of high molecular weight genomic DNA from sweet orange (Citrus sinensis) suitable for subsequent digestion by rarely cutting restriction enzymes and then separation by pulsed-field gel electrophoresis (PFGE). Methods previously described for preparation of plant DNA prior to PFGE involved protoplast isolation, a procedure that can be inefficient and time-consuming for several plant species. Nuclei isolated from plant tissues were embedded into agarose blocks and treated to release DNA, which was cleaved by restriction enzymes and then submitted to PFGE. One gram of fresh leaves gave approximately 15 micrograms of high molecular weight genomic DNA (> 2000 kbp). Within-gel hybridizations were used instead of classical Southern blotting, and the resulting signals were adequate when they were compared with those obtained with DNA prepared from crude ground leaf tissues.     

Extracellular stimulation of epidermal DNA synthesis

The removal of the topmost cell layers of the epidermal stratum corneum by stripping initiates a series of biochemical events which alters the normal homeostatic control of and results in the acceleration of the cell cycle in basal cells which are ten to twenty cell layers removed from the site of stripping. One measure of accelerated events is a stimulation of thymidine incorporation into epidermal DNA at time intervals following stripping. Two peaks of maximal stimulation occur between 12 and 24 hr and 48 and 54 hr after stripping. Stimulation of thymidine incorporation into epidermal DNA by limited stripping is a useful technique for studying the stripping-initiated signal at the stratum corneum and its subsequent translation at the proliferative cell receptor site.     

Kv3.3b: a novel Shaw type potassium channel expressed in terminally differentiated cerebellar Purkinje cells and deep cerebellar nuclei

A two-step hybridization/subtraction procedure was employed to isolate markers for the later stages of Purkinje cell differentiation. From this screen, a novel Shaw potassium channel cDNA (Kv3.3b) was identified that is developmentally regulated. Expression of this channel is highly enriched in the brain, particularly in the cerebellum, where its expression is confined to Purkinje cells and deep cerebellar nuclei. Sequence analysis revealed that it is an alternatively spliced form of the mouse Kv3.3 gene, and that the previously reported Kv3.3 mRNA (Ghanshani et al., 1992) is not expressed in cerebellum. Expression of the Kv3.3b mRNA begins in cerebellar Purkinje cells between postnatal day 8 (P8) and P10 and continues through adulthood, coinciding with elaboration of the mature Purkinje cell dendritic arbor. The timing of expression of Kv3.3b mRNA is maintained in mixed, dissociated primary cerebellar cell culture. These results suggest that the Kv3.3b K+ channel function is restricted to terminally differentiated Purkinje cells, and that analysis of the mechanisms governing its expression in vivo and in vitro can reveal molecular mechanisms governing Purkinje cell differentiation.     

Loss of water from the stratum corneum induces epidermal DNA synthesis in hairless mice

Many clinical studies have shown that low humidity has a deleterious effect on skin, but the mechanisms involved are poorly understood. To clarify the changes that occur in skin, we examined epidermal cell proliferation in mice kept in a dry (relative humidity < 10%) or a moist (relative humidity > 90%) environment. In animals exposed to low humidity, epidermal DNA synthesis started to increase within 12 h, reaching twice the original level, and the increased level was maintained for up to 5 days. The transepidermal water loss (TEWL) of mice kept for 12 h in the dry environment was the same as that of mice kept in the moist environment, but the skin conductance was lower. The increase in epidermal DNA synthesis following exposure to the dry environment was inhibited by topical application of petrolatum. It is concluded that loss of water from the stratum corneum induces epidermal cell proliferation within 12 h, and this change occurs in the absence of apparent cutaneous barrier dysfunction.     

Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220+ population generated a single transient wave of IgM+ IgD+ B cells but failed to generate T cells. In contrast, transfer of the B220- fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.     

An ATP-dependent iron transport system in isolated rat liver nuclei

A concerted translational control is responsible for maintaining an iron level in the cytosol that is both adequate for the synthesis of iron-containing proteins and does not represent a danger to the cell. However, little is known about how iron level is controlled in the nucleus. Nuclei of rat liver take up iron from ferric citrate by a process that is dependent on ATP. This system shares several properties with known P-type ATPases, suggesting that a P-type ATPase in the nuclear membrane is responsible for iron transport. (i) Adenosine 5'-(beta,gamma-iminodiphosphate), a non-hydrolyzable ATP analogue, does not support iron uptake; (ii) the uptake is strongly inhibited by vanadate; (iii) there is an absolute requirement for Mg2+; and (iv) reagents that oxidize SH groups inhibit uptake, and this inhibition can be prevented by dithiothreitol. The energy of activation for the uptake (11.5 kcal/mol) and the Km for ATP (0.4 mM) are similar to values for other known cation transport ATPases. Inhibitors of Na+,K+-ATPase, sarcoplasmic reticulum Ca2+-ATPase, proton V-ATPase, and nuclear Ca2+-ATPase have no effect on uptake. Ferric citrate can be replaced by Fe-ATP as a source of iron for the transport system; however, two other stronger iron chelators, Tiron and desferrioxamine, completely inhibit the uptake. Taken together, these data strongly suggest that an Fe-ATPase, distinct from other known P-type ATPases, is responsible for iron transport in the nucleus.     

Effect of polyene antibiotics on isolated dog kidney nuclei

Effect of amphotericin B and nistation on caryoplasmic proteins, nuclear membrane-bound chromatin (DNPm) and soluble DNP (DNP0) from dog kidney isolated nuclei is studied in vitro. The yield of caryoplasmic proteins from nuclei is found to be increased during incubation with amphotericin B and nistatin, the content of some fraction in caryoplasmic proteins being decreased while their qualitative composition being unchanged. It is found that the treatment of nuclei with amphotericin B contributes the association of DNP particles with nuclear membrane and the increase of protein/DNA and RNA/DNA ratios in DNPm. No such effects are observed in the presence of nistatin. Both antibiotics do not affect protein/DNA and RNA/DNA ratios in DNP0 fraction. Polyene antibiotics are shown to change considerably the composition of acid soluble proteins in DNP0 and DNPm and in non-histone proteins in DNP0, and not to affect the content of lipids and their fatty acid composition in DNPm. The data obtained are of certain value for explanation of the mechanism of toxic effect of polyene antibiotics on animal cells.     

Modulatory action of epidermal growth factor on differentiated human granulosa lutein cells: cross-talk between ligand activated receptors for EGF and gonadotropin

A number of local regulatory factors including polypeptide growth factors like epidermal growth factor (EGF) have been suggested to play an active role within the human ovary. In order to understand the physiology of EGFs action, it is essential to demonstrate and characterize the receptors for this growth factor on ovarian cells which was the aim of this study. We demonstrate using [125I]EGF that specific high affinity sites with Ka for this ligand reaching 2.2 x 10(-9) M for growing cultures of human granulosa-lutein cells and 0.13 x 10(-9) M for the membrane fraction prepared from these cells. Additionally we have identified a 170 kD protein as the EGF receptor with the help of affinity cross linking and immunoblotting procedures. Furthermore, we observed that a pretreatment of granulosa lutein cells with EGF for a short duration (0-30 min) leads to a dose- and time dependent upregulation of the LH-receptor-coupled adenylate cyclase activity. A maximal effect (159 +/- 12% increase compared with untreated cells, P < 0.001, n = 4) was reached at 10-15 min with 10-20 ng/ml EGF. Specific inhibition of the receptor tyrosine kinase activity abolished the observed EGF-induced sensitization of the cyclase activity. Differentiation of granulosa cells in vivo is a prerequisite for ovulation and later transformation into highly differentiated lutein cells, a process depending on the presence of ligands that elevate cAMP production. The observed modulation of the adenylate cyclase by EGF could be a regulatory component for the differentiated status of the granulosa cells during different phases of the cycle.     

Identification of acutely isolated cells from developing rat cerebellum

Immunocytochemical staining was used to identify nerve and glial cells from postnatal rat cerebelli in situ and following tissue dissociation. Purkinje cells were identified using antibodies for the calcium-binding proteins calbindin and PEP19. Purkinje cells isolated during the second postnatal week were 15-20 microns in diameter and relatively abundant and displayed thin perisomatic processes. These features were used to identify Purkinje cells with scanning electron microscopy, which revealed extensive membrane infoldings. Golgi and nuclear cells were identified using antibodies against rat-303 antigen. Pale, nuclear, and Purkinje cells were identified using antibodies for rat-302 antigen. Although staining for rat-302 and rat-303 was weak during the second postnatal week, we were able to identify Golgi and pale cells even after tissue dissociation. Isolated Golgi cells were 8-10 microns in diameter and fewer in number than Purkinje cells and did not counterstain with calbindin antibodies. Isolated pale cells were 8-10 microns in diameter, rare, and resistant to calbindin antibodies. Isolated neurons from cerebellar nuclei were not located with either 302 or 303 staining, suggesting that they remained in the tissue. Golgi-Bergmann cells and astrocytes were identified using antibodies for glial fibrillary acidic protein. Isolated glial cells were 12-15 microns in diameter, more numerous than Purkinje cells, and unstained with calbindin antibodies. With phase-contrast optics, glial cells appeared flatter than neuronal cell types and had acentric nuclei. These results demonstrate that specific cell types in developing rat cerebellum can be identified after acute isolation, which should facilitate analysis of their endogenous properties.