Extended expression of differentiated function in primary cultures of adult liver parenchymal cells maintained on nitrocellulose filters *1I. Induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase

Cr, Savage; Rj, Bonney

doi:10.1016/0014-4827(78)90488-3

Cited by 62 publications

(11 citation statements)

References 16 publications

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“…It has been well documented that inducible P1450 but not P3450 expression appears earlier in development (20,29,41), persists in cultured cells long after P3450 and other forms of P450 are lost (1, 25,36,37,40), and remains in relatively dedifferentiated tumors (33) and preneoplastic nodules (3) after P3450 and other forms of P450 are no longer expressed. The P1450 gene is constitutively expressed and inducible in mouse hepatoma Hepa-1 cultures, whereas P3450 gene expression is completely absent in this cell line (21).…”

Section: Resultsmentioning

confidence: 99%

Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.

Kimura¹,

Gonzalez²,

Nebert³

1986

Mol. Cell. Biol.

170

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Expression of the P1450 and P3450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methykcholanthrene. AU six tissues were shown to have increased P1450 and P3450 mRNA concentrations after treatment with these inducers. P3450 mRNA induction was more sensitive than P1450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P3450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P1450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily paraflel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P1450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P3450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content.In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P3450 gene were at least 10-fold less than those of the P1450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations.Among laboratory animals such as mice, rats, and rabbits, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible P450 gene family comprises two genes: P1450 and P3450 in the C57BL/6N mouse (13); P450c and P450d, respectively, in the rat (38, 39); and form 6 and form 4, respectively, in the rabbit (18, 32). The P1450 and P3450 genes appear to lie adjacent to one another near the Mpi-J locus of mouse chromosome 9 (19), have most likely arisen via gene duplication at least 65 million years ago (24), and are regulated by the aromatic hydrocarbon (Ah) receptor, the gene product of the Ah locus (for a review, see reference 9).Mouse P1450 protein is known to metabolize benzo[a]pyrene better than P3450 protein (31). Similarly, it has been reported that rat P450c protein metabolizes benzo[a]pyrene at least 50 times better than rat P450d protein (35). On the other hand, the rat P450d protein is more important than the rat P450c protein in the metabolism of 2-aminofluorene, 4-aminobiphenyl, and two promutagenic pyrolysis chemicals to active mutagenic and carcinogenic intermediates (23

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Section: Resultsmentioning

confidence: 99%

Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.

Kimura¹,

Gonzalez²,

Nebert³

1986

Mol. Cell. Biol.

170

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“…Since it has been shown that other cells grow on cellulose nitrate membrane filters (17) and that viruses other than poliovirus adsorb to such filters (8), we expect that the methodological approach described here may be applicable to a wide range of cells and viruses. Moreover, since most nonenveloped viruses suspended in aqueous solutions adsorb to cellulose nitrate filters (8), it may be possible to use the methodological approach described here to detect viruses in lowtiter viral suspensions.…”

Section: Discussionmentioning

confidence: 99%

“…Animal cells attach to membranes with rough surfaces and pores, such as those of cellulose nitrate, and multiply until they cover the whole surface of the membrane (17). The present study was initiated on the assumption that cells would not only cover the rough surface of the cellulose nitrate membranes but also penetrate through the pores.…”

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confidence: 99%

A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

Papageorgiou

Mocé-Llivina

Christodoulou

et al. 2000

Appl Environ Microbiol

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We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N؉ membranes after the preparations were transferred.

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“…The collagen gel/nylon mesh support system represents an alternative to the floating collagen gel (8) and Millipore filter (27) substrata and, as such, provides a number of advantages over these two as well as the more conventional plastic (10,28) and collagen-coated plastic surfaces (29) used to support rat hepatocytes in primary cultures. § Unlike the floating collagen gel, the collagen coated/nylon mesh does not shrink with time in culture, it is easier to handle than the thin flexible gels, and it allows for the rapid removal and separation of the hepatocytes from their collagen substratum with collagenase.…”

Section: Discussionmentioning

confidence: 99%

Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes.

Sirica¹,

Richards²,

Tsutsumi³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

210

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Hepatocytes from adult rats were maintained in primary culture for up to 10-13 days on nylon meshes coated with a thin layer of rat tail collagen gel. Their ultrastructu're closely resembled that of the liver parenchymal cell in vivo, but hepatocytes in late culture exhibited a pronounced buildup of microfilaments beneath their apical cell surface. Hepatocytes in earl and late cultures secreted albumin, transferrin, and al-acid glycoprotein into the medium; they exhibited a 7-to 10-fold induction of tyrosine aminotransferase activity by dexamethasone; and they expressed an alkaline phosphatase that was similar to that of normal rat liver with respect to its inhibition by the liver enzyme inhibitor L-homoarginine. In addition, the hepatocytes in culture demonstrated phenotypic changes characteristic of fetal liver parenchymal cells. These changes, which paralleled an increase in DNA synthesis, included the expression of and linear increase in the activity of the fetal liver cell enzyme y-glutamyl transpeptidase, an increased production of a1-fetoprotein, and a c ange in the substrate specificity of fructose-bisphosphate aldolase to that of the fetal liver isozyme.It is well-established that, in vivo, hepatocytes from adult rats express fetal liver cell characteristics during hyperplasia and hepatocarcinogenesis (1, 2). Examples include expression by the hepatocyte of the enzyme y-glutamyl transpeptidase (3-5), production of a-fetoprotein (6), and the synthesis of fetal isozymes of enzymes such as fructose-bisphosphate aldolase (7). The functional significance and biochemical regulation of these transitions as they relate to the proliferative state are for the most part unknown and are relatively difficult to study in vivo. We now report a system for maintaining adult rat hepatocytes in primary culture and provide evidence for their rapid transition to a more fetal-like state characterized by the production of fetal proteins and an increase in hepatic DNA synthesis.MATERIALS AND METHODS Reagents. Leibovitz (L-15) tissue culture medium, penicillin/streptomycin mixture, and fetal calf serum were purchased from GIBCO. Hi/Wo/Ba medium (10 times concentrated) was obtained from International Scientific Industries (Cary, IL). Insulin, collagenase (type 1), L-y-glutamyl-p-nitroanilide, glycylglycine, p-nitrophenyl phosphate, D-fructose 1,6-bisphosphate, D-fructose 1-phosphate, and a-glycerophosphate dehydrogenase-triosephosphate isomerase were purchased from Sigma. Dexamethasone phosphate was purchased from Merck, Sharp & Dohme; NADH from Boehringer Mannheim; and L-(+)-homoarginine from Aldrich. y-Glutamyl-4-methoxy-2-naphthylamide was obtained from Vega-Fox Biochemicals (Tucson, AZ), and [methyl-3H1thymidine (40-60 Ci/mmol) was purchased from New England Nuclear.Preparation of Collagen Gel/Nylon Mesh Substratum. Swiss nylon monofilament mesh fabric (Nitex HC3-253) was purchased from TETKO (Elmford, NY). Circular meshes were cut from the fabric to fit the bottoms of 100 X 20 mm tissue culture dishes (Falcon). Mes...

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Extended expression of differentiated function in primary cultures of adult liver parenchymal cells maintained on nitrocellulose filters *1I. Induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase

Cited by 62 publications

References 16 publications

Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.

Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.

A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes.

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