Organ culture of adult mouse intestine. I. Morphological results after 24 and 48 hours of culture.

Explants of adult mouse intestine have been maintained in organ culture for 24 to 48 hr. The best results have been obtained with a mixture of DMEM-HEPES medium and NCTC-135 enriched with 10% fetal bovine serum. The morphology of the mucosa is well preserved at the light and electron microscopic level: absorbing cells exhibit an increase in secondary lysosomes; goblet cells and Paneth cells remain active; numerous mitoses are observed in the crypts; and vigorous re-epithelization takes place on the margin of the explants.     

Organ culture of adult mouse intestine

Summary Explants of adult mouse intestine have been maintained in organ culture for 24 to 48 hr. The best results have been obtained with a mixture of DMEM-HEPES medium and NCTC-135 enriched with 10% fetal bovine serum. The morphology of the mucosa is well preserved at the light electron microscopic level: absorbing cells exhibit an increase in secondary lysosomes; goblet cells and Paneth cells remain active; numerous mitoses are observed in the crypts; and vigorous re-epithelization takes place on the margin of the explants. This work was supported by a research grant from the Medical Research Council of Canada (to J. S. H.) and partly by the F.C.A.C. Québec (to J. S. H.).     

Organ culture of adult mouse intestine

Summary Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase is measured during the first 24h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.This work was supported by research grants from the Medical Research Council of Canada (J.S.H.) (D.M.)Mr. Chabot is a recipient of a studentship from the Medical Research Council of CanadaD. Ménard, Ph.D. is chercheur-boursier from the CRSQ     

Organ culture of adult mouse intestine. III. Behavior of the proteins, DNA content and brush border membrane enzymatic activities.

The proteins, the DNA content and the brush border membrane enzyme activities of adult mouse intestinal explants have been measured during a 24-hr organ culture. These activities were not modified in comparison with the controls at the beginning of the culture. The activity of the enzymes secreted in the medium during the 24-hr culture was equal to the activity present in the explants at the start of the culture. These results show that several metabolic functions of the intestine are fully preserved in organ culture.     

Organ culture of adult mouse intestine IV. Stimulation of glucose-6-phosphatase in vitro.

Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase in measured during the first 24 h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.     

Organ culture of chicken cecum: Morphologic and physiologic observations after 24 and 48 h of culture

Summary Cecum from chickens, 4 wk old, can best be maintained for 24 h in a serum-free organ culture system using Trowell T8 medium agar sheet at 25°C. As determined by light microscopy as well as scanning and transmission electron microscopy, mucosal architecture involving classic ultrastructure of chicken cecal mucosa was preserved. Protein content of cecal explants did not change up to 48 h of culture. DNA content of cecal explants did not change up to 24 h of culture but decreased significantly to two-thirds of control in 48 h of culture. Based on the morphologic and physiologic findings, it became evident that this organ culture system using Trowell T8 medium at 25°C can be successfully used as an in vitro experimental model for as long as 24 h. The organ culture system could be a useful tool, from the structural integrity of ceca observed in this study, in investigating mucosal function and mucosal response to drugs, carcinogens, trophic factors, and pathogens.     

Organ culture of adult Rat and mouse tracheal epithelium: I. Ultrastructure following various culture periods

Summary Electron microscopic studies of adult rat and mouse tracheal epithelium maintained in organ culture for a period of up to 6 days were performed. In specimens cultured for 60 minutes no conspicuous micromorphological alterations could be observed. Following culture periods from 1–6 days the number of cilia in some of the ciliated cells was reduced while their structure and the other ultrastructural details of the epithelial cells were preserved. In specimens cultured for 5–6 days some additional alterations could be noticed: polymorphism of mitochondria, increased number of lysosomes, appearance of intracellular vacuoles, exhaustion of goblet cells and disappearance of granulated mast-cell like cells in the rat tracheal epithelium.I want to thank Miss J. Selbmann and Mrs. S. Kolassa for technical help and Mr. H. Wagner for preparing the micrographs; I am indebted to Dr. D. Kerjaschki and to Mr. H. Hörandner for performing preparations for scanning electron microscopy and to Mr. P. Scholze (Österreichische Studiengesellschaft für Atomenergie, Institut für Metallurgie, Abteilung Fremdkörperphysik) for preparing the scanning electron micrograph.This work was supported by the Fond zur Förderung der wissenschaftlichen Forschung: Project 2099.This paper is dedicated to Prof. Bargmann on the occasion of his 70th birthday.The author wishes to express her appreciation to Prof. Stockinger for suggestion and advice.     

DNA synthesis in monkey kidney cellular culture, hematoporphyrin sensitized and He--Ne laser irradiated.

He--Ne laser irradiation for 1-45 J/cm2 has four actions upon monkey kidney cell cultures: a) a stimulation of nuclear DNA synthesis after 20 J/cm2; b) a diminution of replication after higher dose (45 J/cm2); c) inhibition of nuclear DNA synthesis and d) a marked cytotoxic effect. At lower radiation fluences (1-5 J/cm2) the influence on the biosynthesis of DNA is not significant.     

Mitotic activity and cell degeneration in the mouse thymus over a period of 24 hrs

Summary The mitotic and pyknotic indices were calculated over a period of 24 hrs in four different zones of the mouse thymus (Balb/C). Counts were based on 1 micron sections of Epon-embedded tissue. This technique gives a more accurate mitotic index than has been calculated in the past because mitotic figures in the prophase stage are more easily recognizable. Mitotic as well as pyknotic index showed a distinct diurnal rhythm in the subcapsular zone, the rhythm being less distinct towards the medulla, where no significant diurnal fluctuations were demonstrated. It was shown that the index variations could be correlated to significant variations in the mitotic time.     

Endoglin overexpression modulates cellular morphology, migration, and adhesion of mouse fibroblasts.

Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Endoglin glycoprotein is a component of the transforming growth factor type beta (TGF-beta) receptor system which is highly expressed by endothelial cells, and at lower levels on fibroblasts and smooth muscle cells, suggesting the involvement of these lineages in the HHT1 vascular dysplasia. Overexpression of endoglin in mouse NCTC929 fibroblasts led to decreased migration in chemotactic and wound healing assays, as well as changes in the cellular morphology. When plated on uncoated surfaces, endoglin transfectants formed intercellular clusters, endoglin being not specifically localized to the cell-cell junctions, but homogenously distributed on the cellular surface. Although the expression of alpha5beta1 integrin and of an activation epitope of beta1 integrin were unchanged, a polyclonal antibody to alpha5beta1 integrin was able to inhibit cluster formation, suggesting the involvement of integrin ligand/s. In fact, coating with fibronectin, laminin, or an RGD-containing 80 kDa fragment of fibronectin were able to prevent the cellular clustering. Furthermore, synthesis of plasminogen activator inhibitor 1 (PAI-1), and to a weak extent that of fibronectin, were inhibited in endoglin transfectants. Thus, the presence of endoglin in mouse NCTC929 fibroblasts is associated with reduced production of certain extracellular matrix (ECM) components, which might explain their altered morphology, migration and intercellular cluster formation.     

Regulatory factors produced by lymphocytes. II. Inhibition of cellular DNA synthesis associated with a factor inhibiting DNA polymerase alpha activity.

Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce (3)H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase alpha in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from alpha-lymphotoxin (alpha-LT), is completely inactivated by heating at 70 degrees C for 15 min. This treatment does not destroy alpha-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of alpha-LT inhibits thymidine incorporation into HeLa cells without causing cell death. alpha-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.     

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.     

Inhibition of DNA replicative synthesis and DNA repair synthesis in human and mouse cells in culture by cigarette smoke condensate fractions.

Twelve cigarette smoke condensate fractions were tested for their ability to inhibit replicative DNA synthesis and DNA excision repair synthesis in cultures of human fibroblasts and Swiss mouse embryo cells. None of the fractions showed any specificity for the inhibition of DNA repair and, in general, repair synthesis was less sensitive to inhibition than was replicative synthesis. There was some correlation between the inhibitory action of the various fractions and their activity in bioassays performed in other laboratories, including $\text{in vitro}$ cell transformation and bacterial mutagenicity. In most cases, DNA synthesis in the human cells was more sensitive to inhibition than it was in the mouse cells. The specific compounds in the condensate fractions which are responsible for their activity have not been identified.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and alpha-fetoprotein production.

The effects of exposure of rat hepatocytes in primary maintenance culture to chemical carcinogens has been studied with respect cytotoxicity and alterations in mitotic index, unscheduled DNA synthesis and alpha-fetoprotein (AFP) production. All compounds tested produced cytotoxicity. Increases in mitotic index and unscheduled DNA synthesis and the production of AFP were observed after treatment of the cells with the carcinogens but not after treatment with the non-carcinogenic isomers. These increases were dose-dependent and depended on the time of exposure and the time incubated postexposure. The patterns of the increase in mitotic index and AFP production after cessation of carcinogen exposure were very similar, with the increase in mitotic index occurring slightly before that for the AFP production and it is suggested from this and other data that the production of AFP is dependent on the generation of a cell species functionally distinct from the non-dividing hepatocytes. It is also suggested that measurement of unscheduled DNA synthesis in conjunction with that of AFP production in cultured hepatocytes may be useful as part of a screening programme for chemical carcinogens.     

Ruthenium(II) polypyridyl complexes: synthesis and studies of DNA binding, photocleavage, cytotoxicity, apoptosis, cellular uptake, and antioxidant activity

Two ruthenium (II) complexes [Ru(dmb)2(APIP)](ClO4)2 (APIP=2-(2-aminophenyl)imidazo[4,5-f?][1,10]phenanthroline, dmb=4,4'-dimethyl-2,2'-bipyridine; 1) and [Ru(dmb)2(HAPIP)](ClO4)2 (HAPIP=2-(2-hydroxyl-4-aminophenyl)imidazo[4,5-f?][1,10]phenanthroline; 2) were synthesized and characterized. DNA binding was investigated by electronic absorption titration, luminescence spectra, thermal denaturation, viscosity measurements, and photocleavage. The DNA binding constants for complexes 1 and 2 were 4.20 (±0.14)×10(4) and 5.45 (±0.15)×10(4) M(-1). The results suggest that these complexes partially intercalate between the base pairs. The cytotoxicity of complexes 1 and 2 was evaluated by MTT assay. Cellular uptake was observed under fluorescence microscopy; complexes 1 and 2 can enter into the cytoplasm and accumulate in the nuclei. Apoptosis and the antioxidant activity against hydroxyl radicals (?OH) were also explored.