The response of normal and polyoma virus-transformed BHK-21 cells to exogenous purines

Adenosine (10 μM) stimulates the initial growth rate of BHK/21 cells seeded at low but not high density in monolayer culture; it does not affect final cell density or permit growth in agar. In labelling experiments With tritiated thymidine, adenosine also increases the response of quiescent cells to low concentrations of serum. Dialysis of serum to remove oxypurines only marginally reduces its effect on quiescent cell labelling or growth, indicating that BHK/21 cells are able to synthesise purines. The response of quiescent cells to 5% serum is inhibited by high MW (2 × 10⁶) dextran sulphate at 2 μg per milliliter. Low MW dextran sulphate (30,000) and heparin at 20 μg per milliliter produce the same effect. Exogenous adenosine (10 μM) prevents this inhibition. Many other purine derivatives replace adenosine for all the above activities but xanthine is completely inactive in all. It, therefore, appears that nucleotide synthesis is a necessary function of these compounds. The growth of cells of a polyoma-virus-transformed BHK/21 line in monolayer is not stimulated by exogenous purine, though their colony-forming ability in agar is increased five-fold. The stimulating effects of exogenous purines on normal BHK/21 cells and the absolute requirement for them in the presence of polyanions is discussed in relation to possible mechanisms of growth control.     

The translational requirement for complete La Crosse virus mRNA synthesis is cell-type dependent.

The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells.     

Phenotypic variation in human HepG2 hepatoma cells: alterations in cell growth, plasma protein synthesis and heme pathway enzymes

1. Growth rates, morphology, plasma protein synthesis and the level of heme pathway enzymes were examined in six sublines of HepG2 cells obtained from various laboratories. 2. Five sublines represented by G2a display the known characteristics of HepG2 cell type, including morphology, plasma protein synthesis and an increase in delta-aminolevulinic acid (ALA) dehydratase activities in response to Me2SO treatment. 3. In contrast, cells of the G2f subline failed to secrete significant quantities of plasma proteins. There was also no increase in ALA dehydratase activity following Me2SO treatment. These findings suggest that G2f cells represent a variant of HepG2 cells with an altered phenotype.     

Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone.     

Cell proliferation and cell cycle dependent changes in the methylthioadenosine phosphorylase activity in mammalian cells

The objective of this study is to investigate the activity of methylthioadenosine phosphorylase (MTA-Pase) in mammalian cells stimulated by serum to proliferate and during their cell cycle. A direct correlation between growth rate and MTA-Pase activity in chinese hamster ovary (CHO) cells was observed. High MTA-Pase activity was observed during the exponential growth phase followed by a low enzyme activity during plateau phase of growth. To understand whether the fluctuations in the enzyme activity was cell cycle dependent, initially the activity of MTA-Pase was studied in plateau phase (G0) CHO cells as they synchronously go into S phase upon plating in fresh medium. The MTA-Pase activity in G0 cells before initiation of growth was 10.3 n.mol/mg protein/30'. A peak activity of 16.0 n.mol/mg/30 min was found at 12 hr after stimulation of proliferation by serum. These results indicate a peak MTA-Pase activity between 10-12 hr after stimulation of proliferation coinciding with the initiation of DNA synthesis. The activity of the enzyme slowly decreased as the cells completed their DNA synthesis. To understand whether these fluctuations are cell cycle specific, HeLa cells were synchronized in different phases and MTA-Pase activity was studied. The specific activities of the enzyme were 2.76, 2.99, 3.97, 3.28 and 3.65 n.moles/mg/30 min. in mitosis, early G1, late G1, S and G2 phases of the cell cycle respectively. These results indicate that MTA-Pase activity peaks in late G1 phase before the initiation of DNA synthesis, similar to the polyamine biosynthetic enzymes and might play a role in the initiation of DNA synthesis by salvage of adenine into nucleotide pools.     

Studies on the nature of protease-induced growth stimulation in normal and transformed BHK cells.

In the presence of growth-limiting serum concentrations trypsin displays mitogenic activity on actively-growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease-induced growth stimulation. Previous work strongly suggested that the trypsin active-site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity. Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma-transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin-like enzymes in the growth and transformation of BHK cells.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

UDP-galactose 4-epimerase: a key enzyme in exopolysaccharide formation by Lactobacillus casei CRL 87 in controlled pH batch cultures

AIMS: To evaluate the relationship between exopolysaccharide (EPS) production and the sugar nucleotide biosynthetic enzymes in Lactobacillus casei CRL 87 under optimum growth conditions for polymer formation: controlled pH on galactose or glucose. Studies with an EPS mutant were carried out to determine the key enzymes in EPS synthesis under the above culture conditions. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method, while the activities of the biosynthetic enzymes were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. An environmental pH of 5.0, using galactose as carbon source, markedly improved not only polymer production and yield but also, cell growth and lactic acid production. Analysis of the activities of the EPS precursor-forming enzymes revealed that polysaccharide synthesis was correlated with uridine-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase under these growth conditions. CONCLUSIONS: EPS synthesis by Lact. casei CRL 87 was considerably improved at a controlled pH of 5.0 with galactose as carbon source, and was correlated with the activity of UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase. The results obtained with the wild-type and EPS- strains suggest that UDP-galactose 4-epimerase plays an essential role in EPS formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Unravelling the key enzymes involved in EPS biosynthesis under optimum culture conditions for polymer production provides important information for the design of strategies, via genetic engineering, to enhance polysaccharide formation.     

Modulation of lysosomal enzyme levels in cultured cells: effects of alterations in cell density, balanced growth, and endocytosis

Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β-N-acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

Growth effects and metabolism of arginine in stationary and rapidly-dividing sugarcane cell suspensions

The effect of 300 μ M arginine on growth of sugarcane cell suspensions was studied. Cells transferred to defined media in the stationary growth stage showed a greater requirement for exogenous arginine than cells similarly transferred in the rapidly dividing stage. Cell arginine levels, rates of arginine synthesis, and enzymes of arginine synthesis all decreased in cells entering the stationary stage. It is concluded that stationary stage cells are deficient in their ability to synthesize arginine and are therefore dependent upon an exogenous supply to resume growth in fresh media.     

Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.     

Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase. More efficient biocatalysis for synthesis of alpha-Gal epitopes

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.     

Isolation of bovine retinal pigment epithelial cells using adhesion to agarose: demonstration of cellular and regional heterogeneity.

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.     

Effect of exogenous DNAses on various stages of DNA replication in microorganisms

Effect of DNAase 1 on DNA synthesis and cell division was studied in microorganisms deficient in some stages of DNA replication initiation. The DNA synthesis induced by exogenous DNAase was found to be a replicative origin since it was registered from the "origin" of chromosomal replication under the conditions of initiation of proteins functioning. Stimulation of DNA synthesis in bacterial cells having mutations in DNA B and DNA G genes by DNAase 1 indicates that exogenous DNAases participate in replicative fork during the DNA synthesis.     

Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog

Baby hamster kidney (BHK) cells transformed with Rous sarcoma virus, RS-BHK cells, demonstrate a 2.5-fold increase in the activity of N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155), and this increase in activity appears to be specific for this enzyme. By contrast, a lectin-resistant BHK cell line selected for its ability to grow in high levels of L-phytohemagglutinin, LP3.3, is characterized by a specific decrease in its GlcNAc-T V activity. To test if these alterations in the apparent Vmax of GlcNAc-T V are due to changes in the efficiency of populations of enzymes in RS-BHK and LP3.3 cells compared to the parental BHK cells, we have compared the kinetic properties of the enzymes from these three sources. The Km constants observed for both the sugar nucleotide donor (UDP-GlcNAc) and two synthetic trisaccharide acceptors were indistinguishable. The Vmax values toward three synthetic acceptors were also determined first for the BHK GlcNAc-T V, and they varied by over 5-fold. When these values were measured for the variant and transformed cell enzymes, however, similar 5-fold differences were still observed, although the absolute values for these acceptors were all higher or lower for the RS-BHK and LP3.3 enzymes, respectively. In addition, we have synthesized a deoxygenated analog of the specific GlcNAc-T V acceptor, beta GlcNAc(1,2) alpha Man(1,6) beta ManOR, where the reactive 6'-OH group has been removed, and the resulting trisaccharide was found to be a competitive inhibitor of the enzyme. The Ki for this inhibitor was near 70 microM for the GlcNAc-T V from all three sources. These kinetic comparisons demonstrate that the enzymes from the three cell types have kinetically indistinguishable active sites. These results suggest that the differences in the apparent Vmax values among the cell types are most likely due to alterations in the number of active molecules rather than in the modulation of either their catalytic activities or specificities.     

Apolipoprotein E inhibits serum-stimulated cell proliferation and enhances serum-independent cell proliferation

Independently of its role in lipid homeostasis, apolipoprotein E (apoE) inhibits cell proliferation. We compared the effects of apoE added to media (exogenous apoE) with the effects of stably expressed apoE (endogenous apoE) on cell proliferation. Exogenous and endogenous apoE increased population doubling times by 30-50% over a period of 14 days by prolonging the G(1) phase of the cell cycle. Exogenous and endogenous apoE also decreased serum-stimulated DNA synthesis by 30-50%. However, apoE did not cause cell cycle arrest; both apoE-treated and control cells achieved equivalent saturation densities at 14 days. Further analyses demonstrated that exogenous and endogenous apoE prevented activation of MAPK but not induction of c-fos expression in response to serum growth factors. Endogenous (but not exogenous) apoE altered serum concentration-dependent effects on proliferation. Whereas control (non-apoE-expressing) cell numbers increased with increasing serum concentrations (1.6-fold for every 2-fold increase in serum), apoE-expressing cell numbers did not differ as serum levels were raised from 2.5 to 10%. In addition, in low serum (0.1%), apoE-expressing cells had elevated DNA synthesis levels compared with control cells. We conclude that apoE does not simply inhibit cell proliferation; rather, the presence of apoE alters the response to and requirement for serum mitogens.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types : Effect of growth-promoting hormones and nutrients

The ability of prostaglandin F_2α (PGF_2α) and other prostaglandins to stimulate the initiation of DNA synthesis in quiescent cultures of various mouse fibroblastic cell types has been investigated. PGF_2α was found to be more effective than the other prostaglandins. Most cell types, with the exception of BALB/c 3T3, responded to PGF_2α. Addition of PGF_2α in combination with insulin resulted in a synergistic increase in the proportion of cells synthesizing DNA. The effect of nutrients on the stimulation of the initiation of DNA synthesis has been examined in detail; it was found that Swiss 3T3 cells showed a requirement for hypoxanthine and vitamin B₁₂ whereas Swiss 3T6 cells demonstrated a stringent requirement for vitamin B₁₂ only. The effect of prostaglandin precursors, synthetic analogues of the prostaglandin endoperoxides and inhibitors of prostaglandin synthesis was also examined in two cell types. The effect of PGF_2α was compared with that of two polypeptide growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in Swiss 3T6 cells grown in 0.0025% (v/v) serum. In combination with insulin each of these three growth factors stimulated the initiation of DNA synthesis in approximately the same number of cells.