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.     

Effect of cell density on the inhibition of tumor cell attachment and nucleic acid synthesis by selenite

The effect of cell density on the sensitivity of tumor celles to selenite has been examined. The inhibitory effect of selenite on cellular DNA and RNA synthesis was significantly greater in higher density cultures of HeLa cells and A2780 ovarian tumor cells. High-density cells were also more sensitive to the inhibitory effect of selenite on cell attachment. This difference could not be accounted for by a higher intracellular level of glutathione, since there was no significant difference between the cells at high or low density. The high-density cells were found to take up more selenium per cell during the exposure period; the resulting higher level of intracellular Se could explain their greater sensitivity to selenite. This hypothesis is supported by the observation that DNA synthesis in nuclei isolated from high-density cells did not exhibit higher sensitivity to inhibition by selenite than synthesis in nuclei isolated from low-density cells.     

Effect of X rays on virus production and DNA synthesis in adenovirus-infected cells.

The effect of X rays on the process of type 10 adenovirus infection in HeLa cells was tested. CPE development and virus infections titer were lower in the irradiated cells, while CF titer remained unchanged. The rate of DNA synthesis, as measured by 3H-thymidine incorporation, was higher in the infected cells and radiosensitivity of the process was higher in the infected cells compared with uninfected controls.     

Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits RNA synthesis in L-929 cells (mouse fibroblast line) and HeLa cells (human epitheloid carcinoma line) within 2 min of addition of the compound to the medium. By removing DRB from the medium, the inhibition is promptly and completely reversed after treatment of cells for as long as 1 h or even longer. The inhibitory effect of DRB on the overall rate of RNA synthesis is similar in L and HeLa cells and is markedly concentration-dependent in the low dose range (5-20 muM or 1.6-6.4 mug/ml), but not as higher concentrations of DRB. At a concentration of 12 muM, DRB has a highly selective inhibitory effect on the synthesis of nuclear heterogenous RNA in L cells. At higher concentrations, there is also inhibition of 45 S ribosomal precursor RNA synthesis, but at all concentrations the effect on heterogeneous RNA synthesis in L cells in considerably greater than that on preribosomal RNA synthesis. In HeLa cells, too, DRB has a selective effect on heterogeneous RNA synthesis, but quantitatively the selectivity of action is somewhat less pronounced. In both L and HeLa cells, the inhibition of synthesis of nuclear heterogeneous RNA is incomplete even at very high concentrations of DRB (150 muM). Thus, while DRB is a selective inhibitor of nuclear heterogeneous RNA synthesis, not all such RNA synthesis is sensitive to inhibition. It is proposed that messenger precursor RNA synthesis may largely be sensitive to inhibition by DRB. In short-term experiments, DRB has no effect on protein synthesis in L or HeLa cells. DRB has a slight to moderate inhibitory effect on uridine uptake into L cells and a moderate to marked effect on uptake of uridine into HeLa cells.     

Mitogen-stimulated phospholipid synthesis in normal and immune-deficient human B cells

Eight patients with common variable panhypogammaglobulinemia were shown in the in vitro Ig biosynthesis assay to have defective B cell responses to pokeweed mitogen (PWM). Phospholipid synthesis was assessed in the B cell plus monocyte fraction (MB) and irradiated T cells (T*) of patients and paired normal controls. Cell populations were studied separately and in the four possible combinations (1:1), with and without PWM, to reveal the effect of cell interactions. At 16 to 20 hr the mean stimulation index (SI) +/- standard error for MB cells alone was 1.01 +/- 0.02 for eight patients and 0.99 +/- 0.02 for the paired normals; the T* cell SI was 1.25 +/- 0.04 for patients and 1.28 +/- 0.05 for normals. Combinations of normal MB cells with normal T* cells showed significantly higher SI when compared with the combinations of normal MB cells with patient T* cells (p less than 0.005). However, the combination of patient MB cells with patient T* cells and the combination of patient MB cells with normal T* cells were not significantly different in SI (0.05 less than p less than 0.1). Isolation of patient and normal B cells, T* cells, and monocytes after the choline pulse showed that patient B cells gave a higher SI with normal T* help than with patient T* help. Of greatest interest is the finding that patient B cells that were defective in PWM-stimulated Ig production nevertheless showed a phospholipid synthesis response to PWM in the normal range, suggesting that the maturation defect in these B cells occurs later than the phospholipid synthesis acceleration step, or on a different pathway.     

Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.     

The effect of methylnitronitrosoguanidine on DNA synthesis in human and animal cells. Inhibition of DNA synthesis in asynchronous and synchronous cultured cells

Methylnitronitrosoguanidine (MNNG) is reported to inhibit DNA synthesis in intact human cells, in the cells from patients with ataxia telangiectasia (AT) or the cells from two rodent species. DNA synthesis in different cell lines exhibits varying sensitivity to MNNG inhibitory effect. 4-5-fold higher concentrations of MNNG are required for 50% inhibition of DNA synthesis in AT cells or in field vole cells as compared with the concentration required for human cells or Chinese hamster. The different compactness of two chromatin fractions might possibly result in lower sensitivity of DNA synthesis in heterochromatin to MNNG-induced inhibition as compared with the sensitivity of euchromatin. The genetic expression of AT defect on the cellular level is supposed to be connected with changes in supramolecular packaging of chromatin in interphase nuclei.     

Effect of elastase on prostacyclin synthesis in aortic smooth muscle cells

The effects of elastase on prostacyclin biosynthesis in cultured rat aortic smooth muscle cells were investigated. Prostacyclin is the major product formed from arachidonic acid by aortic smooth muscle cells. When intact cells were incubated with elastase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident. However, the addition of elastase directly to the cell-free homogenates did not show any effects on prostacyclin biosynthesis. The maximal effect of elastase on the stimulation of prostacyclin biosynthesis without any cellular damage was observed at a concentration of 50 unit/ml elastase. Elastase also caused a marked release of arachidonic acid. At higher concentrations of elastase (75-100 units/ml), the release of arachidonic acid and prostacyclin synthesis was observed, but, at these concentrations of elastase, cells were slightly damaged. On the other hand, the releases of prostacyclin and arachidonic acid were markedly enhanced, when cells were preincubated with elastase (1 unit/ml) for 3 days. These results indicate that elastase, even at low concentrations, causes the releases of arachidonic acid and prostacyclin, especially when aortic smooth muscle cells are pre-treated with elastase.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

High ambient temperature directly decreases milk synthesis in the mammary gland in Saanen goats

The mammary gland is a privileged organ for mammals. Because of their high capacity for milk synthesis, dairy ruminants have been distributed throughout the world. In tropical areas, dairy animals face high ambient temperatures (HTa). The indirect effect of HTa on milk synthesis is mediated in part by a reduction in feed intake. The current experiment focused on the direct natural effect of HTa on mammary function. Multiparous Saanen goats were used in this study. The physiological responses for HTa were evaluated from the control period during the winter and from the natural HTa during the summer. Milk samples were collected for isolation of the goat milk cells to study the expression of the β-1,4 galactosyltransferase (β-GALT1), Akt, and heat shock protein 70 (HSP70) genes. Although goats in the summer maintained rectal temperature and plasma cortisol levels similar to those observed in the winter, the higher respiratory rate and lower feed intake and milk yield (MY) from the goats in the summer indicated that the goats in the summer were exposed to a higher degree of HTa. This was supported by the significantly higher level of plasma glutathione peroxidase (GPX) activity. Moreover, the relative expression levels of β-GALT1 and Akt were not different. The relative expression of HSP70 during the summer was significantly higher than what was observed in cells isolated in the winter. In conclusion, the HTa effect on MY during the summer was related to its indirect effect on feed intake. The direct HTa effect might be related to HSP70 gene expression in goat milk cells and to plasma GPX activity. However, the natural HTa did not affect the expression of Akt or β-GALT1.     

5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells.

Porphyrin synthesis and iron accumulation was stimulated by exogenous 5-aminolevulinic acid (ALA) in uninduced Friend erythroleukemic cells (FELC). Uroporphyrin and protoporphyrin were the major intermediated precursors produced. All porphyrin types were conjugated to protein insoluble cellular components and could be extracted only by methanol sulfuric acid esterification. Heme content of the uninduced FELC was increased 6-fold in the presence of 5 x 10(-4) M ALA. As a consequence, the synthesis of the minor murine hemoglobin component was preferentially induced, an effect similar to that expressed by exogenous hemin. Addition of exogenous ALA to 0.5% DMSO-induced cells increased total hemoglobin synthesis with a higher efficiency of the minor hemoglobin. The endogenous synthesis of porphyrin from exogenous ALA was markedly reduced by hemin. Uroporphyrin, coproporphyrin, protoporphyrin and heme were equally repressed, indicating an inhibitory effect of hemin on ALA dehydrase and urosynthetase activities. In addition, hemin repressed [3H]leucine incorporation into protein by uninduced cells. Incubation of uninduced cells in culture medium without serum in the presence of hemin blocked their protein synthesis activity, whereas addition of serum exerted a protective effect on living FELC.     

Inhibition of protein synthesis by a tryptic polypeptide of Clostridium perfringens type A enterotoxin

The biological activity of Clostridium perfringens enterotoxin can be tested more precisely and with a much higher sensitivity by using the inhibition of protein synthesis by Vero cells, rather than the guinea pig skin test. Tryptic peptides of the enterotoxin produced in the presence of different concentrations of sodium dodecyl sulfate (0-1%) have been tested for biological activity (Vero cells) and inhibitory effect on cell-free protein synthesis (rabbit reticulocyte lysate). A fraction of tryptic peptides, about 16,000 daltons, was able to inhibit the cell-free protein synthesis, while the native enterotoxin had no such effect. The 16 kDa fraction had, however, lost the ability to disrupt the Vero cells (normal biological activity). It is probable that the enterotoxin has the double function (A and B chain), known from several other toxins, confined in its single polypeptide chain.     

Design,synthesis, and evaluation of quinazoline T cell proliferation inhibitors

We report here on a class of quinazoline molecules that inhibit T cell proliferation. The most potent compound N-p-tolyl-2-(3,4,5-trimethoxyphenyl)quinazolin-4-amine (S101) and its close analogs were found to inhibit the proliferation of T cells from human peripheral blood mononuclear cells (PBMC) and Jurkat cells, with IC₅₀ in the sub-micromolar range. The inhibitor induced G2 cell cycle arrest but did not inhibit IL-2 secretion. The anti-proliferative effect correlated with inhibition of the tyrosine phosphorylation of SLP-76, a molecular element in the signaling pathway of the T cell receptor (TCR). The inhibitor restrained proliferation of lymphocytes with much higher potency than non-hematopoietic cells. This new class of specific T cell proliferation inhibitors may serve as lead molecules for the development of agents aimed at diseases in which T cell signaling plays a role and agents to induce tolerance to grafted tissues or organs.     

Regulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast.

The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib2 mutants) with the blocked second reaction to flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoro-methyl-10-(beta-hydroxyethyl)izoalloxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.     

Effect of age and dietary restriction on protein synthesis by isolated kidney cells

The influence of age and food restriction on kidney protein synthesis was studied in Fischer F344 rats. The rate of total protein synthesis by suspensions of kidney cells declined 60% between 4 and 31 months of age. The rate of protein synthesis by kidney cells isolated from 19-month old rats fed a restricted diet (60% of diet consumed by rats fed ad libitum) was 45% higher than the rate of protein synthesis by kidney cells isolated from 19-month old rats fed ad libitum. The excretion of protein in the urine was measured to assess the effect of the age related decline in protein synthesis on kidney function. A dramatic increase in proteinuria was observed with increasing age, and rats fed the restricted diet excreted significantly less protein in the urine than rats fed ad libitum.     

Effect of interferon-alpha on immunoglobulin synthesis by human B cells

We have investigated the effect of human recombinant interferon-alpha (IFN-alpha) on mitogen-induced immunoglobulin (Ig) production by peripheral blood mononuclear cells from normal individuals. Low concentrations (1 to 100 IU/ml) of IFN-alpha enhanced pokeweed mitogen-stimulated Ig production. In contrast, high concentrations of IFN-alpha (10(5) IU/ml) suppressed pokeweed mitogen-induced Ig production. Irradiation of T cells did not ablate the high dose suppression, indicating that suppression was not due to a radiation-sensitive T cell. Kinetic experiments revealed that IFN-alpha needed to be added to 10 day cultures within the first 72 hr for either enhancement or suppression to be noted. Preincubation of purified B cells with IFN-alpha suppressed Ig production as completely as when unfractionated mononuclear cells were incubated with IFN-alpha. On the other hand, preincubation of T cells or monocytes with IFN-alpha had no effect on subsequent Ig production in reconstituted mononuclear cell cultures. Mitogen-induced proliferation of purified B cells was not affected by IFN-alpha at any concentration, but Ig production by purified B cells stimulated with Staphylococcus aureus Cowan I or anti-mu and B cell differentiation factors responded to IFN-alpha with low concentration enhancement and high concentration suppression. Studies of Ebstein-Barr virus-transformed B cell lines showed that IFN-alpha caused a similar effect on the CESS line as on peripheral blood B cells, with low dose enhancement and high dose suppression of Ig production. Thus one IFN-alpha effect is to modulate Ig production, and this appears to be a direct effect on B cells. Combined with the data in the accompanying paper, the effects of IFN-alpha on B cell function are similar in vivo and in vitro.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Enhancement of DNA synthesis of human epithelial carcinoma cells by acidic fibroblast growth factor

Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.     

Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans

The effect of in situ immobilization of Gluconobacter oxydans on a novel carrier material in a repeated-fed-batch operated packed-bed bubble-column bioreactor for the production of the fine chemical dihydroxyacetone was investigated experimentally. The carrier material were biocompatible, durable, coated Ralu-rings. The coating was a porous silicone matrix with satisfactory wetting characteristics. Settling of cells was relatively rapid. The cells were protected from abrasion caused by shear forces. A sufficiently high oxygen supply rate to the immobilized cells was provided due to the high oxygen permeability of the silicone matrix. The immobilized biomass was estimated to be about 65% of the total biomass contained in the bioreactor after 18 days of operation. The observed space-time yield was approx. 76% higher compared to a similar process which was performed without an optimized fermentation medium. Compared to previous experiments with a trickle-bed bioreactor, the space-time yield was approx. 3.7 times higher. A typical time course of the immobilization process was observed: after an induction phase, a transition phase followed which later on gave way to a nearly linear accumulation phase. A stationary phase with regard to the amount of immobilized active cells, however, was not reached. Hence, a higher bioreactor performance than observed could be expected at longer operation times.