The chemistry of thiamine: Studies on the dimerization of thiazolium salts

In this communication we report on our studies into the previously undetected dimerization chemistry of thiazolium salts. Thiazolium salts with electron-withdrawing substituents, such as 3,4-dimethyl-5-ethoxycarbonylthiazolium iodide, yield acid- and oxygen-sensitive ethylenic dimers under conditions originally used to detect the dimerization of 3-methylbenzothiazolium iodide. The 5-ethoxycarbonyl-4-methyl-3-phenylmethylthiazolium and 5-(2-O-triphenylmethyl-hydroxyethyl)-4-methyl-3-phenylmethylthiazolium bromides yield stable rearranged dimers, rather than the labile ethylenic dimers, under identical conditions. 4-Methyl-5-(2-hydroxyethyl)-3-phenylmethylthiazolium bromide and thiamine hydrochloride yield rearranged dimers which were isolated as their N,O-ketal derivatives when these salts were heated in aprotic solution in the presence of DBN and K₂CO₃, respectively. Rearrangement of the ethylenic dimer of 3-phenylmethylbenzothiazolium bromide to 2-(benzothiazol-2-yl)-2,3-diphenylmethylbenzothiazoline (J. Baldwin, S. E. Branz, and J. A. Walker (1977) J. Org. Chem. 42, 4142) demonstrates that rearranged dimers of these thiazolium salts are produced via a mechanism involving 1,3-sigmatropic rearrangement of intermediate ethylenic dimers. Based on literature precedent we argue that this dimerization chemistry demonstrates the nucleophilic carbene nature of C-2 deprotonated thiazolium salts in aprotic basic solution.     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photosynthetic electron transport in thylakoid preparations from two marine red algae (Rhodophyta).

Thylakoid membrane preparations active in photosynthetic electron transport have been obtained from two marine red algae, Griffithsia monilis and Anotrichium tenue. High concentrations (0.5-1.0 M) of salts such as phosphate, citrate, succinate and tartrate stabilized functional binding of phycobilisomes to the membrane and also stabilized Photosystem II-catalysed electron-transport activity. High concentrations (1.0 M) of chloride and nitrate, or 30 mM-Tricine/NaOH buffer (pH 7.2) in the absence of salts, detached phycobilisomes and inhibited electron transport through Photosystem II. The O2-evolving system was identified as the electron-transport chain component that was inhibited under these conditions. Washing membranes with buffers containing 1.0-1.5 M-sorbitol and 5-50 mM concentrations of various salts removed the outer part of the phycobilisome but retained 30-70% of the allophycocyanin 'core' of the phycobilisome. These preparations were 30-70% active in O2 evolution compared with unwashed membranes. In the sensitivity of their O2-evolving apparatus to the composition of the medium in vitro, the red algae resembled blue-green algae and differed from other eukaryotic algae and higher plants. It is suggested that an environment of structured water may be essential for the functional integrity of Photosystem II in biliprotein-containing algae.     

Reversible dissociation of malate dehydrogenase from plants]

It was demonstrated that 0.2 M citric acid (pH 2.5) inactivates highly-purified malate dehydrogenase from tea leaves; the degree of inactivation depends on temperature and time of incubation. The enzyme activity is restored by certain inorganic salts, the degree of reactivation being dependent on pH, ionic strengths of salts and duration of enzyme incubation with both inactivating and reactivating agents. Urea and guanidine hydrochloride also have a reversibly inactivating effect on the enzyme. The degree of inactivation depends on their concentration and incubation time. In the latter case reactivation of enzyme is achieved by dialysis or 20-40-fold dilution of the enzyme preparation. A kinetic study demonstrated that inactivation of enzyme by the above-mentioned agents is due to the enzyme dissociation into 4 catalytically inactive subunits with molecular weights of 17 500 +/- 1000, which under certain conditions are capable of reassociating into an active molecule of enzyme with completely restored native conformation.     

Studies on plant growth-regulating substances XXIX. The plant growth-retarding properties of certain ammonium, phosphonium and sulphonium halides

Wheat, pea and dwarf bean seedlings grown under controlled environmental conditions were used to assess the growth-retardant activity of members of series of chloro-substituted benzyl-, trimethyl- and tri-n-butyl- ammonium bromides. The influence on activity of trialkyl groupings other than tri-methyl and tri-n-butyl was also studied using compounds with the 4-chloro-benzyl ring structure. 3-Chloro- and 4-chloro-benzyltributylammonium bromides were the most effective compounds. The activity was similar to that of 2,4-dichlorobenzyltributylphosphonium bromide (Phosphon-D) and they showed little phytotoxicity. A series of chlorophenoxymethyltributylammo-nium and phosphonium salts were found to have lower activity than the corresponding chlorobenzyl derivatives. Allyldimethylsulphonium bromide retarded the growth of wheat seedlings but, like the aliphatic trimethylam-monium bromides tested, it was only slightly active in the pea-seedling test. The results are considered in relation to the chemical structure of the compounds studied. In particular, the influence of chlorine substitution in the ring of benzyltributylammonium salts on their growth-retardant activity is compared with the effect of similar substitution on the auxin activity of phenoxyacetic acids.     

Synthesis of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-ones and their 2-methylene derivatives as potential spermicidal and microbicidal agents

A series of twenty two derivatives of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one and their 2-methylene derivatives were synthesized from naturally abundant cinchonine (I). Tartarate salts of these compounds were prepared and evaluated for spermicidal activity. The most active compounds (24, 27, 34, 36, and 38) showing potent spermicidal activity were further evaluated against different strains of Trichomonas vaginalis, for antimicrobial activity, in HeLa cell lines for cytotoxicity and against Lactobacillus jensenii for eco-safety. The tartarate of 3-(1-pentyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one (27) was found to be more active than N-9 in spermicidal activity.     

Analysis of the facilitatory effect of the ACTH-(4-9) analog ORG 2766 on active social contact in rats

Pairs of male rats were tested for active social interaction, either in a familiar test arena under low illumination or in an unfamiliar test arena under high illumination conditions. Rats tested in an unfamiliar environment and under high light, spent less time in active social contact than rats tested under familiar, low light conditions. This effect was most pronounced during the first half of the 10 minute test period. Intraperitoneal injections of ACTH-(1-24) and ACTH-(4-10) (50 micrograms/kg) administered 5 minutes before the test decreased, whereas the same dose of the synthetic ACTH-(4-9) analog ORG 2766 increased the time spent in active social contact, when rats were tested under unfamiliar, high light conditions. The effects of ACTH-(4-10) and ORG 2766 were present in the second and first half of the test period respectively. Dose response relationship studies with ORG 2766 showed that 0.5 micrograms/kg of this peptide facilitated social contact under both test conditions and the dose response relation followed an inverted U-shaped curve under the familiar low light condition, but not under the unfamiliar, high light condition. ACTH-(4-10) and ORG 2766 failed to influence active social contact, when administered 30 minutes before the test. The change in social contact by ACTH-(4-10) and ORG 2766 was not accompanied by an alteration in ambulation of the rats. It is concluded that ACTH-(4-10) and ORG 2766 decrease and increase respectively social interaction of pairs of rats. The expression of these effects however, depends on the test and treatment conditions and may be related to the action of brain-born ACTH-like peptides.     

Corticoliberin fragment CRF4-6 modifies rat behavior in opposite ways under stressing and non-stressing conditions

The effects of tripeptide corticoliberin fragment CRF4-6 (icv; 6, 30, and 150 nmol/head) on behaviour of rats were investigated under non-stressing and stressing conditions. CRF4-6 activated rat behaviour under non-stressing conditions (home cage): the duration of locomotion and exploratory behaviour was increased whereas the duration of passive behaviour and sleep was decreased. On the other hand, CRF4-6 suppressed the rat exploratory behaviour under stressing conditions (elevated plus-maze): the duration of non-exploratory behaviour was increased; numbers of rearings and leanings out to open arms were reduced. All observed effects of the tripeptide CRF4-6 were dose-dependent. Behavioural effects of the tripeptide were similar to the well-known action of the whole corticoliberin molecule. Therefore we suggest that CRF4-6 is located in the active part of CRF or it can be a physiologically active corticoliberin derivative.     

Effect of salinity on seed germination,growth and ion content in dimorphic seeds of Salicornia europaea L.(Chenopodiaceae)

The halophyte Salicornia europaea L. is a widely distributed salt-tolerant plant species that produces numerous dimorphic seeds. We studied germination and recovery in dimorphic seeds of Central Asian S. europaea under various salinity conditions. We also tested the effects of various salts on Natand Ktaccumulation during plant development from germination to anthesis under greenhouse conditions. We found good germination（close to control） of large seeds under Na Cl between 0.5 and 2%, Na₂SO₄and 2 Na Cl t KCl t Ca Cl between 0.5 and 3%, and 2Na₂SO₄t K₂SO₄t Mg SO4 between 0.5 and 5%. For the small seeds, we found stimulating effects of chloride salts（both pure and mixed） under 0.5e1% concentrations, and sulfate salts under 0.5e3%. Both types of seeds showed high germination recovery potential. Salt tolerance limits of the two seed types during germination and at the later stages of development were very similar（4e5%）. During plant growth the optimal concentrations of mixed chloride and sulfate salts ranged from 0.5 to 2%. The mechanisms of salt tolerance in the two seed types of S. europaea appear to differ, but complement each other, improving overall adaptation of this species to high salinity.     

Dihydrozeatin: An improved synthesis and resolution of both isomers

(±)-Dihydrozeatin was synthesized in a 3-step synthesis by: (1) a Michael condensation of methyl methacrylate with nitromethane to give (±) - methyl 2 - methyl - 4 - nitrobutyrate, which was (2) reduced to (±) - 4 -amino - 2 - methylbutan - 1 - ol and (3) reaction of the aminoalcohol with 6-chloropurine. Hydrolysis of racemic nitroester gave (±) - 2 - methyl - 4 - nitrobutyric acid, which was resolved by means of (+) - and (-) -α -methylbenzylamine salts. Conversion of the salts to the corresponding methyl esters and subsequent reductions yielded optically active 4 - amino 2 - methylbutan - 1 - ols. Examination of the NMR spectra of the resolved methyl 2 - methyl - 4 - nitrobutyrates in the presence of a chiral shift reagent established their optical purities to be greater than 98%. The specific rotations at 589 nm of theS- (?) andR- (+)- dihydrozeatins derived from optically active butanols were appreciably lower than previously reported. Application of the Drude equation to ORD values from 320 to 589 nm verified the low 589 nm rotations of the dihydrozeatin enantiomers. The biological activities of (R), (S) and (R,S) dihydrozeatins in the betacyanin stimulation assay withAmaranthus parallel the activities found in other cytokinin bioassays.     

Hemocyanin from Tachypleus gigas. I. Oxygen-binding properties

Hemocyanin was prepared from an Asian horseshoe crab, Tachypleus gigas. The hemocyanin was found to be similar to Limulus hemocyanin in the size of native molecules (48-mer) and dissociation under nonphysiological conditions. It also showed the reverse Bohr effect. The O2 affinity of the dissociated monomer was higher than that of the native molecule. Equilibrium O2 binding to T. gigas hemocyanin was studied with special attention to the effect of inorganic ions. Neutral salts decreased the O2 affinity of the associated hemocyanin. In the presence of CaCl2 the strength of the effect was in the order of Na+ greater than Cs+ not equal to K+ for the series of chlorides, and Br- not equal to Cl- greater than SO4(2-) for the series of Na+ salts. A high concentration of CaCl2 (50-500 mM) considerably increased the Hill coefficient. The O2 binding data obtained under various ionic conditions were analyzed by model fitting. The two-state concerted model could be fitted to the data, if the ligand affinity of the states was allowed to vary. Statistical tests of the fitting showed that the hexameric structure can be regarded as the functional unit under physiological conditions.     

Critical balance of electrostatic and hydrophobic interactions is required for beta 2-microglobulin amyloid fibril growth and stability

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.     

Optical resolution by preferential crystallization of 4-methylpiperidinium hydrogen (RS)-phenylsuccinate

Gibbs energy of racemate formation, binary melting point diagram, and ternary solubility diagram suggested that 4-piperidinium hydrogen (RS)-phenylsuccinate [(RS)-4-MP salt] exists in a conglomerate. Appropriate conditions were explored on the basis of free energy of critical nucleation in a supersaturated solution to resolve efficiently (RS)-4-MP salt by preferential crystallization. Successive preferential crystallization of (RS)-4-MP salt in ethanol at 20°C gave (R)- and (S)-4-MP salts of 90–94% optical purities. Optically pure (R)- and (S)-phenylsuccinic acids were obtained by recrystallization of the (R)- and (S)-4-MP salts, followed by treatment of the salts purified with hydrochloric acid. © 1994 Wiley-Liss, Inc.     

A Novel Regioselective Desulfation Method Specific to Carbohydrate 6-Sulfate Using Silylating Reagents

Sulfated primary alcohols and methyl α-D-galactopyranoside 6-sulfate were converted into desulfated and trimethylsilylated alcohols and galactoside, respectively, by treating their pyridinium salts with N,O-bis(trimethylsilyl)acetamide (BTSA) or N,O-bis(trimethylsilyl)triftuoroacetamide (BTSTFA) in pyridine. Sulfated secondary alcohols and methyl galactoside 2-, 3-, and 4-sulfates did not have their sulfates eliminated under similar conditions, indicating that the reaction was specific to the primary hydroxyl groups. Methyl α-galactoside 2-sulfate was preparatively obtained by the BTSA treatment of methyl α-galactoside 2,6-disulfate, indicating that the method is applicable to regioselective desulfation on a preparative scale.     

Glucose production and glycogen cycle enzyme activities in avian liver explants: procedural optimization

1. There are few studies which both describe the in vitro regulation of glycogen metabolism in nonmammalian animals and list methods to optimize assay conditions for these studies. 2. A mechanical tissue chopper was used to obtain 25-65 mg liver explants from 14- to 28-day old domestic turkeys to determine assay conditions (substrates, buffers, time), regulators (metals, salts and hormones) and points of endogenous regulation of glycogenolysis (protein phosphorylation and enzyme activity). 3. High- and low-bicarbonate-based buffers (Earle's balanced salts, EBSS and Hanks' balanced salts, HBSS, respectively) were used to determine buffer effects. 4. Glucose release into the incubation environment was greater in HBSS than EBSS. Adding HEPES to further buffer conditions did not change release rates. Adding bicarbonate to HBSS resulted in release rates similar to EBSS. 5. Calcium increased glycogenolysis in the presence of absence of equimolar concentrations of EGTA; potassium had no effect. 6. Porcine insulin (100 ng/ml) did not inhibit glycogenolysis; however, glucose release was increased by dibutyryl cyclic AMP. A noted catecholamine-induced increase in in vitro glycogenolysis and phosphorylase activity indicates that points of regulation are under phosphorylation-dephosphorylation regulation.     

The copper-molybdenum antagonism in ruminants. I. The formation of thiomolybdates in animal rumen

The formation of thiomolybdates, MoO_xS_4?x^2?(x = 0, 1, 2, or 3), from molybdate and sulphide salts in aqueous media has been studied under conditions which simulate the fluid phase in the rumen. The influences of the sulphide:molybdenum ratio, pH and phosphate levels on the nature of the species formed were investigated. The thiomolybdates, in particular the MoS_4^2? ion, have been implicated as the active intermediates in the widespread molybdenum induced copper deficiency that affects ruminants. The results presented here show that, under physiological conditions, di- and trithiomolybdates will form more readily than tetrathiomolybdate.     

The roles of bile salts in the uptake of beta-carotene and retinol by rat everted gut sacs.

The effects of bile salts, Tween 20 and hexadecyltrimethylammonium-bromide on the uptake of beta-[3H]carotene and [3H]retinol by rat-everted gut sacs were studied in vitro under conditions simulating those present in the intestinal lumen during lipid absorption. 2. Micellar solutions significantly enhanced uptake over emulsions. Maximum uptake occurred at the critical micellar concentration of the bile salts mixture. At higher detergent concentrations beta-carotene uptake declined sharply; retinol absorption remained high. 3. In beta-carotene absorption bile salts functioned not only as micellar solubilizers but also may have been required for interaction with the cell membrane or as a transport carrier. In retinol uptake their primary function appeared only to be micellar solubilization. Both uptake and efflux of substrates were enhanced in bile salt micellar solutions compared to the other detergents. 4. Beta-carotene cleavage and conversion to retinyl esters occurred only in bile salts solutions. Retinol esterification was seen with all detergents. These effects increased as the tri/dihydroxy bile salts ratio was increased. 5. Beta-carotene uptake appeared to be reversible and passive at low concentrations. Retinol uptake was reversible, 7-30 times more rapid, and partially inhibited by 2,4-dinitrophenol at higher concentrations. An energy-requiring step may have been rate limiting.     

Osmotic and specific ion effects on the germination of Prosopis strombulifera

BACKGROUND AND AIMS: Salinity can affect germination of seeds either by creating osmotic potentials that prevent water uptake or by toxic effects of specific ions. Most studies have only used monosaline solutions, although these limit the extent to which one can interpret the results or relate them to field conditions. The aim of this work was to evaluate the germination of Prosopis strombulifera seeds under increasing salinity by using the most abundant salts in central Argentina in monosaline or bisaline iso-osmotic solutions, or in solutions of mannitol and polyethylene glycol. METHODS: Seeds were allowed to germinate under controlled conditions in a germination chamber at 30 +/- 1 degrees C and at 80 % r.h. Salinizing agents were KCl, NaCl, Na(2)SO(4), K(2)SO(4), NaCl + Na(2)SO(4) and KCl + K(2)SO(4) and osmotic agents were polyethylene glycol 6000 and mannitol. Treatments for all osmotica consisted of 0.0, -0.4, -0.8, -1.2, -1.5, -1.9 and -2.2 MPa solutions. KEY RESULTS: The percentage of germination decreased as salinity increased. SO(4)(2-) in monosaline solutions, with osmotic potentials -1.2 MPa and lower, was more inhibitory than Cl(-) at iso-osmotic concentrations. This SO(4)(2-) toxicity was alleviated in salt mixtures and was more noticeable in higher concentrations. K(+) was more inhibitory than Na(+) independently of the accompanying anion. CONCLUSIONS: Different responses to different compositions of iso-osmotic salt solutions and to both osmotic agents indicate specific ionic effects. This study demonstrates that the germination of P. strombulifera is strongly influenced by the nature of the ions in the salt solutions and their interactions. Comparative studies of Cl(-) and SO(4)(2-) effects and the interaction between SO(4)(2-) and Cl(-) in salt mixtures indicate that extrapolation of results obtained with monosaline solutions in the laboratory to field conditions can be speculative.