Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells

We have investigated the extent to which modifications in the essential fatty acid content of mammalian cells can affect prostaglandin production. Swiss mouse 3T3 cells stimulated with the calcium ionophore A23187 produced 1.7 to 7 times more prostaglandin E(2) (PGE(2)) when the cultures were supplemented with linoleic acid. Increases in PGE(2) production as a result of linoleic acid supplementation occurred under all culture conditions except during the first 24 hr after attachment, when prostaglandin production was very high. Arachidonic acid supplementation produced a similar enhancement in the capacity of the cells to produce PGE(2), but no appreciable increase occurred when the cultures were supplemented with oleic acid. The phospholipids of the cells exposed to the linoleate-enriched medium contained 4 times more arachidonic acid and twice as much linoleic acid as compared with the corresponding controls. The choline phosphoglycerides were most highly enriched in arachidonic acid, but 2- to 3-fold increases also occurred in the inositol and ethanolamine phosphoglycerides. When cultures initially enriched with linoleic acid were transferred to an unsupplemented medium, the fatty acid composition as well as the capacity of the cells to produce PGE(2) reverted almost to control values. The amount of exogenous arachidonic acid converted to PGE(2) as measured by radioimmunoassay also was greater when the cells were enriched with linoleic acid. Studies with radioactive arachidonic acid indicated that the distribution of prostaglandin metabolites was not affected appreciably by linoleic acid enrichment. These findings suggest that at least two factors contribute to the increased capacity of the cultures supplemented with linoleate to produce PGE(2). One is enrichment of the phospholipid substrate pools with arachidonic acid. The other is an increased ability of the cells to synthesize PGE(2) from unesterified arachidonic acid, perhaps because the prostaglandin-forming enzymes are more active.-Denning, G. M., P. H. Figard, and A. A. Spector. Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells.     

In vitro translation products specified by the transforming region of adenovirus type 2.

Region 1 DNA sequences (map positions 0 to 11% on the linear adenovirus 2 genome) are expressed both early and late in lytic infection and are required for transformation by the virus. During productive infection six distinct cytoplasmic RNAs are synthesized from this region. These RNAs comprise two families, each consisting of three size classes that share 3' sequences. Region 1 RNA's were purified by hybridization selection, using restriction fragments bound to nitrocellulose membranes, and by size fractionation. The isolated RNAs were then translated in cell-free systems derived from wheat germ and rabbit reticulocytes. The family of RNAs specified by 0 to 4.4 sequences includes two RNAs, which are 12S and 13S in size. These RNAs were partially separated by molecular weight and translated. The 13S RNA produced 53,000-dalton (53K) and 41K peptides, and the 12S RNA synthesized 47K and 35K products. The family of RNAs mapping from 4.4 to 11.0 encodes three separate polypeptides, each of which can be assigned to a specific RNA. A 12K product that comigrates with structural polypeptide IX is synthesized from the 9S RNA as previously reported (U. Pettersson and M. B. Mathews, Cell 12:741-750, 1977). The 13S RNA encodes a 15K polypeptide that corresponds to a 15K polypeptide in infected cell extracts. The 22s RNA encodes a 52K protein distinct from the 0 to 4.4 polypeptides.     

Properties of fetal and adult red blood cell arginase: a possible prenatal diagnostic test for arginase deficiency.

Prenatal diagnosis of inborn errors of metabolism has been possible only if the enzyme affected is expressed in amniotic fluid cells grown in culture. Arginase is essentially undetectable in normal human fibroblasts, amniotic fluid, and amniotic fluid cells but is present in high amounts in red blood cells. It is absent in the red blood cells of patients with liver arginase deficiency. The properties of the enzyme in the red cells of healthy children and adults were compared to those of the enzyme obtained from cord blood red cells of 13--20-week fetuses obtained at hysterotomy. The activities, heavy metal requirements, heat stability, pH optimum, kinetic properties, and reaction with anti-arginase antibody were examined. Both enzyme species were either identical or substantially similar by all criteria. The adult and fetal enzymes are, therefore, probably determined by the same structural gene. Fetal red cells obtained during amniocentesis and amnioscopy should then be a suitable tissue to use to make the prenatal diagnosis of arginase deficiency.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES

Abstract— The total concentrations of vitamin B₆ (B₆) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90 μ mol/l or kg respectively. The mechanisms by which B₆ enters and leaves brain, choroid plexus and CSF were investigated by injecting [³H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [³H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [³H]B₆ readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [³H]B₆ into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [³H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [³H]PIN decreased the amount of [³H]B₆ in brain and also decreased the percentage of [³H]B₆ in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [³H]PIN (1 μ m ) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B₆ from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.     

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells.

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.     

Immunocytochemical localization of casein kinase II during interphase and mitosis

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.     

Brain Micro vessel 12-Hydroxyeicosatetraenoic Acid Is the (S) Enantiomer and Is Lipoxygenase Derived

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.     

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.     

Mutational analysis of the ICP4 binding sites in the 5'' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.