Unearthing novel thiazolidinone building blocks as carboxylic acid bioisosteres.

Aim: Thiazolidinones were prepared as building blocks for the replacement of carboxylic acids. Materials & methods: Chemical syntheses of thiazolidinones were developed. In addition, the drug-likeness of the target compounds was evaluated in silico. Results: The prepared compounds included the novel structure 4; 5-(3-Iodophenylmethylene)-2,4-thiazolidinedione. Conclusion: Exploration of the methods required to synthesize thiazolidinone building blocks was completed. This work allows future generation of bioisosteric analogs of drugs.

Physico-Chemical Conversion of Lignocellulose: Inhibitor Effects and Detoxification Strategies: A Mini Review.

A pretreatment of lignocellulosic biomass to produce biofuels, polymers, and other chemicals plays a vital role in the biochemical conversion process toward disrupting the closely associated structures of the cellulose-hemicellulose-lignin molecules. Various pretreatment steps alter the chemical/physical structure of lignocellulosic materials by solubilizing hemicellulose and/or lignin, decreasing the particle sizes of substrate and the crystalline portions of cellulose, and increasing the surface area of biomass. These modifications enhance the hydrolysis of cellulose by increasing accessibilities of acids or enzymes onto the surface of cellulose. However, lignocellulose-derived byproducts, which can inhibit and/or deactivate enzyme and microbial biocatalysts, are formed, including furan derivatives, lignin-derived phenolics, and carboxylic acids. These generation of compounds during pretreatment with inhibitory effects can lead to negative effects on subsequent steps in sugar flat-form processes. A number of physico-chemical pretreatment methods such as steam explosion, ammonia fiber explosion (AFEX), and liquid hot water (LHW) have been suggested and developed for minimizing formation of inhibitory compounds and alleviating their effects on ethanol production processes. This work reviews the physico-chemical pretreatment methods used for various biomass sources, formation of lignocellulose-derived inhibitors, and their contributions to enzymatic hydrolysis and microbial activities. Furthermore, we provide an overview of the current strategies to alleviate inhibitory compounds present in the hydrolysates or slurries.

Comparative evaluation of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans.

Streptococcus mutans triggers dental caries establishment by two major factors: synthesis of organic acids, which demineralize dental enamel, and synthesis of glucans, which mediate the attachment of bacteria to the tooth surface. Propolis is a natural product that may prevent dental caries. Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, is the most important botanical origin for the production of green propolis (Brazilian propolis) by honeybees. However, whether B. dracunculifolia (Bd) has an anticariogenic effect, like green propolis, remains unknown. Herein, we have made a comparative evaluation of the effects of extracts from green propolis and Bd on the glucan synthesis and acidogenic potential of S. mutans. The inhibitory effects of the extracts on bacterial acid production were evaluated through the potentiometric measurement of pH from bacterial suspensions treated with serial concentrations of both extracts. Besides presenting close inhibitory values at the same concentration range, Bd leaf rinse and green propolis extracts had similar IC(50) values (0.41 and 0.34 mg/ml, respectively). Both extracts produced a bacteriostatic effect on S. mutans cultures at a concentration of 0.40 mg/ml. Estimated inhibitory values of green propolis and Bd leaf rinse extracts on the synthesis of insoluble glucans (IC(50)=12.9 and 25.0 microg/ml, respectively) and soluble glucans (IC(50)=50.4 and 49.1 microg/ml, respectively) were not significantly different from each other at p<0.05. The results demonstrate that Bd leaf rinse and green propolis extracts have similar inhibitory effects on the S. mutans cariogenic factors evaluated herein, and allowed us to suggest that Bd leaves may be a potential source for pharmaceutical products employed for this purpose.

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats.

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [(3)H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3'-sulfonate (Woodward's Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H(+) exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H(+) antiport mechanism.

Fumonisin B(1) induces apoptosis in LLC-PK(1) renal epithelial cells via a sphinganine- and calmodulin-dependent pathway.

Fumonisins are a family of mycotoxins produced by Fusarium moniliforme, which is the most common mold found on corn throughout the world. These compounds are both toxic and carcinogenic for animals, and perhaps humans, with the kidney being the most sensitive organ to fumonisin toxicity. The molecular mechanism of fumonisin toxicity appears to involve disruption of de novo biosynthesis of sphingolipids and accumulation of sphinganine. The goals of this study were to determine whether fumonisin B(1) kills LLC-PK(1) renal kidney epithelial cells by inducing apoptosis and to identify genes affected by sphinganine that mediate fumonisin B(1)-induced cell death. Fumonisin B(1) produced morphological changes (i.e., cell shrinkage, membrane blebbing) and time-dependent increases in DNA fragmentation demonstrating that the toxin induces apoptosis. Simultaneously, fumonisin B(1) blocked sphingolipid biosynthesis and caused accumulation of sphinganine. To further investigate the role of sphinganine in fumonisin B(1)-induced apoptosis, beta-fluoroalanine (betaFA) was used to inhibit serine palmitoyltransferase, which catalyzes an earlier step in the sphingolipid biosynthetic pathway. betaFA blocked sphinganine accumulation and prevented fumonisin B(1)-induced DNA fragmentation, confirming that apoptosis induced by fumonisin B(1) is dependent upon accumulation of sphinganine. To examine gene expression, differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied to RNA isolated after 16 h of exposure to fumonisin B(1). Differential expression in response to fumonisin B(1) of a gene identified as calmodulin has been verified by Northern analysis. Sphinganine appears to mediate the effect because betaFA reduces induction of calmodulin mRNA by fumonisin B(1). Fumonisin B(1) also increases calmodulin protein in a concentration-dependent manner and the calmodulin antagonist W7 blocks fumonisin B(1)-induced DNA fragmentation, supporting a role for calmodulin in fumonisin B(1)-induced apoptosis. In contrast, fumonisin B(1) had no effect on expression of bcl-2 family genes (bax, bcl-2, and bcl-x). These findings demonstrate that fumonisin B(1) kills LLC-PK(1) kidney cells by inducing apoptosis. Further, the results establish a sequence of events for fumonisin B(1)-induced apoptosis involving initial disruption of sphingolipid metabolism and accumulation of sphinganine (or a metabolite), which, in turn, induces expression of calmodulin.

Synthesis and biological evaluation of ceramide analogues with substituted aromatic rings or an allylic fluoride in the sphingoid moiety.

The biological activity of synthetic ceramide analogues, having modified sphingoid and N-acyl chains, as well as fluorine substituents in the allylic position, was investigated in hippocampal neurons. Their influence on axonal growth was compared to that of C(6)-N-acyl analogues of natural ceramides. D-erythro-Ceramides with a phenyl group in the sphingoid moiety and a short N-acyl chain were able to reverse the inhibitory effect of fumonisin B(1) (FB(1)), but not of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), on accelerated axonal growth in hippocampal neurons. Moreover, we demonstrated that a ceramide analogue with an aromatic ring in the sphingoid moiety is recognized as a substrate by glucosylceramide synthase, which suggests that the observed biological effects are mediated by activation of the ceramide analogue via glucosylation. Introduction of a methyl, pentyl, fluoro, or methoxy substituent in the para position of the phenyl ring in the sphingoid moiety yielded partly active compounds. Likewise, substitution of the benzene ring for a thienyl group did not abolish the ability to reverse the inhibition of accelerated axonal growth by FB(1). Both D-erythro- and L-threo-ceramide analogues, having an allylic fluorine substituent, partly reversed the FB(1) inhibition.

The effect of fungal competition on colonization of maize grain by Fusarium moniliforme, F. proliferatum and F. graminearum and on fumonisin B1 and zearalenone formation.

The effect of water activity (0.98, 0.95, 0.93) and temperature (15, 25 degrees C) on fungal growth and toxin production from interactions between isolates of Fusarium moniliforme and F. proliferatum producing fumonisin, and an isolate of F. graminearum producing zearalenone, incubated at the same time on irradiated maize grains were determined in vitro. Populations (CFUs) of F. moniliforme and F. proliferatum were reduced to a greater or lesser extent by the presence of F. graminearum under all conditions tested, while that the presence of F. moniliforme or F. proliferatum had a minor inhibitory effect on fungal populations of F. graminearum. Fumonisin B, production by F. proliferatum was inhibited under all conditions tested, while fumonisin B1 production by F. moniliforme was inhibited at 15 degrees C and enhanced at 25 degrees C in the presence of F. graminearum. The level of zearalenone was not significantly modified in the presence of F. moniliforme and F. proliferatum under the conditions tested.

Induction of the mitochondrial permeability transition in vitro by short-chain carboxylic acids.

We recently reported that acrylic acid (AA) induces the MPT in vitro, which we suggested might be a critical event in the acute inflammatory and hyperplastic response of the olfactory epithelium. The purpose of the present investigation was to determine if induction of the MPT is a general response to short-chain carboxylic acids or if there are critical physical chemical parameters for this response. Freshly isolated rat liver mitochondria were incubated in the presence of varying concentrations of selected carboxylic acids. All of the acids that we tested caused a concentration-dependent induction of the MPT, which was blocked by cyclosporine A. Although the C4 carboxylic acids were slightly more potent than the C5 acids, there was no correlation with the degree of saturation, the octanol/water coefficient (log P), or the dissociation constant (pK(a)) of the acids that we tested. We conclude that induction of the MPT in vitro is a general response to short-chain carboxylic acids having a pK(a) of 4 to 5.

Effect of aromatic short-chain analogues of ceramide on axonal growth in hippocampal neurons.

A series of D-erythro- and L-threo-ceramide analogues was synthesized and investigated for their ability to reverse the inhibitory effects of fumonisin B(1) (FB(1)) on axonal growth in hippocampal neurons. The analogues contained either a C(7) side chain or a phenyl group substituted for the C(13) residue present in naturally occurring ceramides, while the N-acyl chain length was reduced from C(16)-C(24) to C(2)-C(8). D-erythro-Ceramide 18a with a C(7) side chain and an N-acetyl residue and D-erythro-ceramide 20c with an aromatic side chain and an N-hexanoyl residue were most active in reversing the inhibitory effects of FB(1) on axonal growth, although the mechanism remains unclear.

Anti-tumor-promoting activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis Ha et Grushv. (I).

Seven saponins (1-7) isolated from the rhizomes and roots of Panax vietnamensis were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor-promoters (cancer chemopreventive agents). The ocotillol-type saponin, majonoside-R2 (2), which is the major and characteristic constituent of this plant, exhibited a significant inhibitory effect on EBV-EA activation. Furthermore, the cell cycle analysis of 2 on Raji cells was also examined and strong inhibition was observed on the effect of the cell cycle induced by TPA. Compound 2 showed potent anti-tumor-promoting activity in two-stage carcinogenesis tests of mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA or fumonisin B1 as a promoter. Consequently, these results suggest that majonoside-R2 (2) could be a valuable chemopreventive agent against chemical carcinogenesis.

Induction of apoptosis by fumonisin B1 in HT29 cells is mediated by the accumulation of endogenous free sphingoid bases.

Fumonisin B1 (FB1) and aminopentol (AP1) (which is formed by hydrolysis of FB1) are found in corn contaminated with some strains of Fusarium moniliforme. Incubation of HT29 cells (a human colonic cell line) with FB1 or AP1 caused a significant reduction in cell number; AP1 was less potent, with 50 microM AP1 causing the same reduction (ca. 30% after 24 h) as 10 microM FB1. The reduction in cell number reflected increases in DNA fragmentation and the percentage of apoptotic cells. Both FB1 and AP1 caused the accumulation of sphinganine (25- and 35-fold by 10 microM FB1 and 50 microM AP1, respectively); thus, concentrations of FB1 and AP1 that caused comparable reductions in cell number were also similar with respect to elevation of sphinganine, a compound that is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with ISP-1 prevented the elevation in sphinganine, DNA fragmentation, and apoptosis induced by FB1. Therefore, these effects of FB1 on HT29 cells can be attributed to the accumulation of sphinganine. Since consumption of food contaminated with Fusarium moniliforme (Sheldon) exposes colonic cells to these mycotoxins, the possibility that FB1 and AP1 are toxic for intestinal cells in vivo should be evaluated, especially in the light of the recent report (Bhat et al., Clin. Toxicol. 35, 249, 1997) describing intestinal disturbances in humans after consumption of moldy corn and sorghum containing fumonisins.

The role of sphingolipids in the maintenance of fibroblast morphology. The inhibition of protrusional activity, cell spreading, and cytokinesis induced by fumonisin B1 can be reversed by ganglioside GM3.

Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3, the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was approximately 45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB1 blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.

Glutathione and the mitochondrial reduction of hydroperoxides.

Various organic hydroperoxides are reduced when added to rat liver mitochondrial suspensions. Succinate increases the rate and duration of the reductions except for linoleic acid hydroperoxide which appears to inhibit its own reduction. 3-Hydroxybutyrate replaces succinate but other reductants used are less effective. The rate of reduction of tert-butyl hydroperoxide by succinate is not inhibited by cyanide but is partly inhibited if antimycin or rotenone are also added; ATP reverses the antimycin inhibition. Other inhibitors include the uncoupler, carbonyl cyanide p-trifluoromethoxyhydrazone, ADP + Pi, the thiol reagents N-ethylmaleimide and p-hydroxymercuribenzoate and inhibitors of the mitochondrial transport of carboxylic acids. In some cases, the GSH concentration of the mitochondria during the reductions correlates with the reduction rate (e.g. with succinate and after N-ethylmaleimide) but in others it is dissociated. The results suggest that hydroperoxide reduction requires the GSH-glutathione peroxidase pathway but that entry of the oxidants into the mitochondrial matrix is also an energy-dependent step.

Antagonism of anthracene-9-carboxylic acid as screening test for muscle relaxants.

Veratrinic response produced by anthracene-9-carboxylic acid in mice has been used as a model for studying effect of variety of drugs which induce muscle relaxation. None of the drugs listed as general depressants, central muscle relaxants, neuromuscular blocking agents and anti-convulsants were effective even at doses causing 100% loss of righting reflex. Local anaesthetics/direct acting muscle relaxants were effective. This simple model employed in conjunction with Straub's tail test can differentiate various categories of muscle relaxants and their mechanism of action.

[Wet dog shake behavior in normal rates, elicited by benzylideneaminooxycarbonic acid derivatives]. / Benzylidenaminooxy-carbonsäurederivate als auslöser von "wet dog shake" - verhalten bei normalen ratten

Wet dog shake (WDS) behavior in rats, well known as morphine-withdrawal syndrome, could be elicited without concomitant symptoms for the first time chemically in non-morphine-addicted animals. The capability to produce WDS was correlated with a specific chemical structure among the title-compounds. The threshold-dose of the most effective agents was 25-50 mg/kg, rather independent of the mode of application. Maximal response of 10-20 WDS per min and animal were reached after application of 100-200 mg/kg. WDS behavior appeared within the first minutes after dose and lasted up to several hours. Detailed information is given on WDS-action of the substance Sgd 8473 = alpha [(4chlorobenzylideneamino)-oxy]-isobutyric acid and the influence by different pharmacologie agents thereon. Inhibition of WDS was produced by: narcotic analgesics, narcotic antagonists, psychosedativ drugs, yohimbine, dl-amphetamine, cocaine, apomorphine and clonidine. Without influence on WDS were: physostigmine, atropine, ganglionic- or adrenergic-blocking drugs, Dopa, MAO-inhibitors, serotonin- and histamin-antagonists and nonnarcotic analgesics. To some extent chemically induced WDS seemed to be susceptible like precipitated WDS. So Sgd 8473 could be qualified for differentiating narctic analgesics, for a "quasiabstinence" agent in research of dependence mechanisms and for a tool in neuroanatomical studies of the CNS.

Effects of the lonophore A23187 on the blood platelets II. Influence on ultrastructure.

A23187, an ionophore which selectively transports calcium across cell membranes into cytoplasm or releases the divalent cation from intracellular storage sites, was shown in previous studies to stimulate platelet aggregation and the release reaction. The nature of its influence on platelet function suggested that an increase in cytoplasmic calcium ion concentration might be a critical factor linking stimulation to secretion through the platelet contractile mechanism. The present investigation has examined the effects of A23187 in platelet ultrastructure and aggregation after incubation with various concentrations of the drug. Scanning and transmission electron microscopy revealed changes in the form and internal organization of platelets following exposure to A23187 that were identical to those which develop in the cells after exposure to potent agents such as collagen and thrombin. High concentrations of ionophore caused destruction of the platelets on prolonged incubation, while the effects of low concentrations on structure and aggregation reversed completely. Recovered platelets were as sensitive to other aggregating agents as control cells. The findings support the concept that platelets are a form of muscle cell and the internal transformation stimulated by A23187 and other aggregating agents is a manifestation of contractile activity. Inactivation of A23187 by plasma followed by recovery of unaltered appearance indicates that platelets have an active mechanism for reducing the level of cytoplasmic calcium.

Effects of the lonophore A23187 on blood platelets I. Influence on aggregation and secretion.

A23187 is an antibiotic ionophore which transports divalent cations across cell membranes into the cytoplasm and releases cations from intracellular storage sites. The present investigation has studied the influence of A23187 on blood platelet aggregation and secretion. A23187 added to stirred C-PRP produced concentration-dependent aggregation and release of (14)C-serotonin. Calcium ions potentiated the response of platelets in washed suspensions to A23187, but they were not required for ionophore-induced aggregation or release. N-ethylmaleimide and agents which increase the level of cyclic 3',5'-adenosine monophosphate (cAMP) in platelets were effective inhibitors of A23187-induced aggregation and secretion.Platelets incubated with (45)Ca(++) before addition of A23187 increased their content of isotope by 50% within 45 seconds after exposure to the ionophore. The results indicate that an increase in the cytoplasmic concentration of calcium ions from intracellular sources may be the critical event in triggering platelet contraction and the release reaction. Increased concentrations of cAMP may inhibit the platelet response to A23187 and other aggregating agents by stimulating a calcium extrusion pump.