Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response.

The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA.     

Inhibition of interleukin 1 synthesis by tenidap: a new drug for arthritis

Tenidap is a new antiarthritic drug of novel chemical structure. This study shows the effects of tenidap on the in vitro synthesis of interleukin 1 (IL-1). IL-1 production by murine peritoneal macrophages was induced either by stimulation with lipopolysaccharide (LPS) or by phagocytosis of zymosan. With either stimulus, tenidap inhibited IL-1 production as measured by a quantitative competitive IL-1 receptor binding assay. Approximately 20 ng/mL of IL-1 was produced by 10(6) macrophages in response to LPS and about half that amount was produced in response to zymosan. Fifty percent inhibition of IL-1 production by tenidap was found at 3 microM for both stimuli. Using goat anti-IL-1 alpha and Western blot analysis, the appearance of intracellular 34 kDa pro-IL-1 alpha was inhibited by tenidap down to 3 microM. Tenidap decreased [35S]Met incorporation into cellular protein at 30 microM but not at 10 or 3 microM, indicating selectivity for IL-1 inhibition relative to total protein synthesis. Because tenidap inhibited IL-1 induction by both zymosan and LPS, it must act subsequently to receptor triggering. As the appearance of IL-1 was inhibited both intracellularly and extracellularly, the primary drug effect cannot be on secretion.     

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

Modulation by propranolol of the uptake of ethidium bromide by rat submandibular acinar cells exposed to a P2X(7) agonist or to maitotoxin

We have compared the formation of pores in rat submandibular acinar cells in response to 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and maitotoxin. Bz-ATP (100 microM) permeabilized the cells to ethidium bromide. The uptake of ethidium increased to 29+/-1% of maximal uptake in 10 min. DL-Propranolol (300 microM) inhibited the Bz-ATP-induced uptake of ethidium bromide by 40% without affecting the P2X(7)-gated cation channel. The inhibitory effect of DL-propranolol on the formation of pores by Bz-ATP was reproduced by D-propranolol, an optical isomer with very poor beta-blocking activity. Tenidap, an antiinflammatory drug, enhanced the permeabilization in response to Bz-ATP. Propanolol inhibited the response to tenidap plus Bz-ATP. The effect of propranolol was reproduced by labetolol, a beta-adrenergic antagonist with membrane-stabilizing properties, but not by atenolol, which blocks beta-adrenergic receptors but has no effect on the stability of the membrane. In the presence of extracellular calcium, maitotoxin also increased the uptake of ethidium bromide. Tenidap had no effect on this response, which was delayed by propranolol. In conclusion, we have shown that propranolol, in a range of 10-300 microM, inhibits the pore-forming activity of the P2X(7) receptor without affecting the opening of the cation channel coupled to this receptor. This inhibition is not related to its beta-adrenergic blocking activity but rather to its membrane-stabilizing properties. Propranolol also delays the uptake of ethidium bromide in response to maitotoxin. This is in agreement with the current view that P2X(7) agonists and maitotoxin share a common pore.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

Inhibition by actinomycin D of ribosomal RNA synthesis in the presynthesis period of the mitotic cycle of Chinese hamster cell cultures]

Using radioautographic technique actinomycin D at a concentration of 0.08 mug/ml was shown to inhibit selectively ribosomal RNA (rRNA) synthesis in monolayer cultures of Chinese hamster cells. The treatment with actinomycin D of cells synchronized by mitotic selection in the beginning of the G1 period causes a delay in the onset of DNA synthesis. However, a similar treatment in the late G1 period does not prevent cells from entering the S-period. The same effect has been produced by 9 mug/ml lucanthone, another inhibitor of rRNA synthesis. The experiments demonstrate a pronounced difference in cell reaction to the depression of rRNA synthesis in early and late G1 period. The data imply that the formation of rRNA, essential for the initiation of DNA synthesis, is accomplished in the first half of the G1 period, while part of rRNA has been already formed in the previous cycle.     

Effects of prostaglandins E1, E2, and F2alpha on the growth of leukaemia cells in culture.

Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine, uridine and leucine. At concentrations of 50 and 25 mug per ml, there was significant inhibition of thymidine and uridine incorporation, but not of leucine, PGF2alpha showed significant inhibition of thymidine and uridine incorporation but not leucine incorporation, in all 3 concentrations studied (100, 50, and 25 mug/ml). The ability of the cells to form colonies in soft agar was significantly inhibited by PGE1 and PGE2 at concentrations as low as 1-8 mug/ml. For F2alpha, however, a concentration as high as 56mug/ml was required to show inhibitory effect, but at 1-8 mug/ml it was found to be stimulatory.     

In vitro lipolytic effect of bovine pituitary diabetogenic protein.

Bovine diabetogenic protein has been further purified by gel filtration yielding a fraction (Mr 25 000--28 000) having increased diabetogenic and in vitro lipolytic activity. Using rat epididymal fat pads, this fraction was shown to be lipolytic at concentrations as low as 1--10 mug/ml. The in vitro lipolytic effect was unaffected by the nutritional state of the animals, was not potentiated by dexamethasone, could be demonstrated in the presence and absence of glucose and was not mediated by alpha- and beta-adrenergic receptors. A lag phase of greater than 1 h was observed before diabetogenic protein induced lipolysis occurred, suggesting that protein synthesis might be involved. Cycloheximide (10 mug/ml), added initially, prevented the diabetogenic protein-induced lipolysis. This direct effect of the purified protein on adipose tissue helps explain the elevation of free fatty acids seen when bovine diabetogenic hormone is administered in vivo and suggests that this anterior pituitary protein may be a new lipid-mobilizing hormone.     

Pyrimido-pyrimidine modulation of EGF growth-promoting activity and p21ras expression in rat mammary adenocarcinoma cells

RA 233, a pyrimido-pyrimidine analogue developed originally as an antiplatelet agent, has reduced the incidence of tumor metastases in clinical trials. However, in animal tumor models antimetastatic therapy using RA 233 has been inconsistent. We therefore tested RA 233 for additional effects, such as its direct action on tumor cells. Using the rat 13726NF mammary adenocarcinoma tumor system, low, nontoxic concentrations of RA 233 had pleiotropic and differential effects on two 13762NF tumor cell clones. The growth of MTC cells (low spontaneous metastatic potential) was not affected by low concentrations of RA 233 (50 microM) or epidermal growth factor (EGF) (up to 10 ng/ml) for 3 days in 0.5-10% fetal bovine serum. In contrast, MTLn3 (high spontaneous metastatic potential) cell cultures maintained for 3 days in low (0.5-1%) serum in the presence of 1.25-10 ng/ml EGF doubled in cell numbers compared with control cultures, and addition of 50 microM RA 233 abrogated the growth-stimulatory effect of EGF. The inhibitory effect of RA 233 on MTLn3 cells was dose dependent and not due to cell toxicity as determined by cell viability, cell growth, and colony formation properties after drug removal. In addition, incubation of MTLn3 cells with 50 microM RA 233 resulted in an increase of p21ras protein expression, whereas there was no effect on the level of p21ras in identically treated MTC cells or when either clone was treated with 10 ng/ml EGF. The results suggest that among the heterogeneous effects of RA 233 on tumor cells, modulation of growth factor responses and regulatory molecules may be important.     

Mouse limb bud cells respond to retinoic acid in vitro with reduced growth.

Retinoic acid (RA) has dramatic effects on the pattern of developing and regenerating vertebrate limbs. These effects are considered to result from RA-induced changes in the positional identity of limb cells, and involve the formation of extra structures. Whether the growth required to form the supernumerary parts of the pattern is a primary effect of RA treatment or a secondary effect that follows after a change in positional identity is not at present known. In this paper we have investigated the effects of RA treatment on the growth of cells from anterior and posterior halves of mouse limb buds in vitro. We observed that under our culture conditions, limb bud cells treated with 1 nM to 1 microM RA (0.3 ng/ml to 300 ng/ml) continue to grow but do so at a significantly slower rate than control cultures. There is a maximum inhibition of growth (50% of controls) between 10 nM and 100 nM RA, which corresponds to the measured range of concentrations of RA in vivo. Our observation of a significant decrease in growth rate over a wide range of RA concentrations is consistent with comparable reports of growth inhibition for a large number of other cell types in vitro as well as with the observation that exogenous RA inhibits blastemal growth in amphibians during the period of exposure to RA. We propose that the effects of RA on growth, either enhancement in vivo or reduction in vitro, can be seen as consequences of the ability of RA to alter positional identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen radical scavenging capacity of phenolic and non-phenolic compounds in red and white wines

The aim of the present study was the evaluation of the antioxidant content in phenolic and non-phenolic extracts of ten wine samples, trying to elucidate the potential role of unusual antioxidant compounds. Samples of wines processed from red and white grapes (Vitis vinifera L.), deprived of the volatile fraction at low temperature and buffered at physiological pH, were fractionated by C₁₈ into two fractions: FR1 and FR2. Non-phenolics, such as tartaric, malic, lactic, and succinic acids; glucose; fructose; and glycerin were mainly found in FR1, while polyphenols were present exclusively in FR2. Peroxyl radical quenching was assayed by the ORAC method, while superoxide and hydroxyl radical scavenging activity were assayed by electron paramagnetic resonance. In the ORAC and superoxide assays, most of the activity was found in FR2, while in hydroxyl radical assay, the activity was found in FR1. Model solutions were used to attribute a role to the single compounds in the evaluation of wine’s ROS scavenging capacity: the ORAC and superoxide anion scavenging effects were mainly attributed to the polyphenols, averaging 94.8%, with some contribution from glycerin, particularly in white wines. Unexpectedly, the main chemical responsible for hydroxyl radical scavenging activity was glycerin (56.1%), with the polyphenols scavenging at 18.1%.     

Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.     

Screening of antiradical and antioxidant activity of monodesmosides and crude extract from Leontice smirnowii tuber.

Leontice smirnowii is a member of the Berberidaceae family. In the current study we investigated the possible antiradical and antioxidant activity of the monodesmosides (MLS) and crude extract (CELS) of Leontice smirnowii using different antioxidant tests: 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, scavenging of superoxide anion radical-generated non-enzymatic system, ferric thiocyanate (FTC) method, reducing power, hydrogen peroxide scavenging and metal chelating activities. Experiment revealed that MLS and CELS have an antioxidant effect concentration-dependently. Total antioxidant activity was performed according to FTC method. At the 30mug/ml concentration, the inhibition effects of MLS and CELS on peroxidation of linoleic acid emulsion were found to be 95.3% and 95.6%, respectively. On the other hand, percentage inhibition of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox were found to be 98.2%, 98.5%, 84.0% and 87.9% inhibition of peroxidation of linoleic acid emulsion, respectively, at the same concentration. In addition, MLS and CELS had effective DPPH radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, reducing power and metal chelating activities. Also, these various antioxidant activities were compared with BHA, BHT, alpha-tocopherol and trolox which were accepted as references antioxidants.     

Effect of fibroblast growth factor on the division and fusion of bovine myoblasts

The effect of fibroblast growth factor (FGF) on the rate of proliferation and fusion of bovine myoblast has been examined. Addition to the cultures of 0.1 mug-1 mug/ml of FGF stimulates the rate of proliferation and delays the fusion of primary cultures of bovine myoblasts cultured in 10% serum. Final cell densities reached in the presence of 0.1 mug/ml of FGF were fivefold higher than in controls; with 1 mug/ml, they were 10-fold higher. Increases in cell density were paralleled by increases in acetylcholine receptor sites as measured by the binding of 125I-alpha-bungarotoxin. Both fusion and the appearance of acetylcholine receptor sites were delayed in the presence of FGF. Growth hormone, insulin and testosterone, which have been reported to be mitogenic for rat and chick embryo myoblasts, did not have significant effects on DNA synthesis in bovine myoblasts when compared to the FGF. Conversely, FGF did not stimulate the proliferation of chick embryo myoblasts, indicating that it is not active in all vertebrate species.     

Effect of Rifamycins and Related Antibiotics on the Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Vaccinia Virus Particles

A number of compounds related to rifampin which act as expected in the Escherichia coli system have been tested for their ability to inhibit the vaccinia particle deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro. Some compounds are inactive even at concentrations of 500 mug/ml, others are able to produce partial inhibition, and others strongly inhibit the enzyme activity at 150 mug/ml or less. The inhibition, where present, operates immediately but appears to be at least partially reversible. At least one compound which is without effect against bacterial RNA polymerase is a potent inhibitor of the viral RNA polymerase. As the enzyme activity of rifampin-resistant mutants of vaccinia virus is inhibited to the same extent as that of the wild type, the observed in vitro effect on vaccinia virus RNA polymerase is not identical with the in vivo effect specifically directed against a vaccinia-specified protein.     

Interstrand cross-linking of DNA by FK317 and its deacetylated metabolites FR70496 and FR157471

FR900482 (1) and FR66979 (2) are structurally novel natural products isolated by Fujisawa in 1987 and have been shown to be highly potent antitumor antibiotics structurally related to the mitomycins. Studies on the mode of action have established that these new agents form covalent DNA interstrand cross-links both in vitro and in vivo as a result of the reactive mitosene intermediate generated upon bioreductive activation. Semisynthetic analogues such as FK973 (3) and FK317 (4) were developed in the search for potentially superior clinical candidates. Although FK317 has been shown to be a potent compound, to date no direct evidence of DNA interstrand cross-link sequence specificity has been reported. In this study, DNA interstrand cross-links were generated by treatment of a synthetic duplex DNA substrate with FK317 (4) and its deacetylated metabolites FR70496 (5) and FR157471 (6). Analysis by gel electrophoresis revealed the formation of orientation isomers displaying electrophoretic mobility vastly greater than the mobilities of those generated from FR900482 (1). Despite these differences, it was established by Fe(II)-EDTA footprinting that FK317 (4) as well as 5 and 6 forms DNA interstrand cross-links within the expected 5'CpG3' step, clearly demonstrating that the phenolic hydrogen in 1 and 2 is not a prerequisite for efficient DNA interstrand cross-linking by the FR class of compounds.     

In Vitro Antiviral Activity of Mycophenolic Acid and Its Reversal by Guanine-Type Compounds

With the agar diffusion test and BS-C-1 cells, mycophenolic acid was found to give a straight-line dose-response activity in inhibiting the cytopathic effects of vaccinia, herpes simplex, and measles viruses. Plaque tests have shown 100% reduction of virus plaques by mycophenolic acid over drug ranges of 10 to 50 mug/ml and virus input as high as 6,000 plaque-forming units (PFU) per flask. Back titration studies with measles virus inhibited by mycophenolic acid have indicated that extracellular virus titers were reduced by approximately 3 logs(10) and total virus was reduced by 1 log(10). The agar diffusion test system lends itself readily to drug reversal studies. Mycophenolic acid incorporated into agar at 10 mug/ml gave 100% protection to virus-infected cells. Filter paper discs impregnated with selected chemical agents at concentrations of 1,000 mug/ml (20 mug per filter paper disc) were placed on the agar surface. Reversal of the antiviral activity of mycophenolic acid was indicated by virus breakthrough in those cells in close proximity to the filter paper disc. Chemicals showing the best reversal of the antiviral activity of mycophenolic acid were guanine, guanosine, guanylic acid, deoxyguanylic acid, and 2,6-diaminopurine. The reversal of antiviral activity was confirmed by titrations of virus produced with various amounts of both mycophenolic acid and guanine present and by isotope tracer methods with uptakes of labeled uridine, guanine, leucine, and thymidine in treated and nontreated, infected and noninfected cells as parameters. All antiviral effects of mycophenolic acid at 10 mug/ml could be reversed to the range shown by untreated controls by the addition of 10 mug/ml of those chemicals exhibiting reversal activity.     

Limitations of the Use of 5-Fluorouracil as a Selective Agent for the Isolation of Leptospirae

It has been shown that some component of Fletcher medium is able to annul the inhibitory action of 5-fluorouracil (400 mug/ml) on bacteria other than leptospirae. The most likely ingredient which can be implicated in this context appears to be beef extract.     

Effects of Actinobolin on Growth and Some Metabolic Activities of Cariogenic Streptococci In Vitro and In Vivo

Actinobolin, a known inhibitor of protein synthesis, has been shown not to interfere selectively with acid production or dextransucrase activity in a cariogenic streptococcus when the antibiotic is added to a concentration of 500 mug/ml. It has also been shown that actinobolin does not alter the total in vivo flora of the oral cavity of the rat when tested in a rat caries model system. A culture of cariogenic streptococci, adapted to in vitro growth in the presence of 1 mg of actinobolin per ml, has also been isolated.