A multivalent lacto-N-fucopentaose III-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective.

A multivalent lacto-N-fucopentaose (LNFP) III-lysyllysine conjugate was observed to decompact preimplantation mouse embryos. Decompaction was not obtained with free oligosaccharides (LNFP II and III), nor with multivalent LNFP II-lysyllysine or chitotriose-lysyllysine conjugates. These results suggest a role for X hapten recognition during compaction and suggest further that X hapten valency may play a key role in modulating this developmental process.

Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label.

1-Nitro-2-phenylethene (beta-nitrostyrene, 1) which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817-818), was demonstrated in this work to be an irreversible inhibitor of beta-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. Kcat values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 x 10(-4) M) ranged from 1.67 x 10(4) S-1 after 30 min of preincubation to <0.07 x 10(4) S-1 after 180 min preincubation. For compound 2 (9.5 x 10(-5) M) Kcat values ranged from 2.70 x 10(4) S-1 following 30 min preincubation to 1.15 x 10(4) S-1 after 180 min of preincubation; the changes in Km(app), however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.

TNF-alpha receptors simultaneously activate Ca2+ mobilisation and stress kinases in cultured sensory neurones.

The cytokine tumour necrosis factor-alpha (TNF) has been implicated in autoimmune diseases and may play an indirect role in activation of pain pathways. In this study we have investigated the possibility that TNF directly activates cultured neonatal rat dorsal root ganglion (DRG) neurones and provides a signalling pathway from cells in the immune system such as macrophages to sensory neurones. Expression of TNF receptor subtypes (TNFR1 and TNFR2) on sensory neurones was identified using immunohistochemistry, fluorescence-activated cell sorting analysis and RT-PCR. Biochemical and immunocytochemical analysis showed that TNF activated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) but not p42/p44 MAPK. TNF treatment evoked transient Ca2+-dependent inward currents in 70% of DRG neurones. These TNF-evoked currents were significantly attenuated by ryanodine or thapsigargin or by inclusion of BAPTA in the patch pipette solution. Responses were also evoked in subpopulations of cultured DRG neurones by human mutant TNFs that cross-reacted with rat receptors and selectively activated TNFR1 or TNFR2 subtypes. TNF-evoked transient increases in [Ca2+]i were also detected in 34% of fura-2-loaded DRG neurones. The link between TNF receptor activation and Ca2+ release from stores remains to be elucidated. However, responses to TNF were mimicked by sphingolipids, including sphingosine-1-phosphate, which evoked a transient rises in [Ca2+]i in a pertussis toxin-insensitive manner in fura-2-loaded DRG neurones. We conclude that distinct receptors TNFR1 and TNFR2 are expressed on cultured DRG neurones and that they are functionally linked to intracellular Ca2+ mobilisation, a response that may involve sphingolipid signalling.

Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

Saccharin induces changes in adenylate cyclase activity in liver and muscle membranes in rats.

The non-nutritive sweetener, saccharin, was found to stimulate significantly the activity of adenylate cyclase in membranes derived from skeletal muscle of rat. Sodium saccharin enhanced adenylate cyclase activity in a dose-related manner, and this activation appeared to be dependent on the presence of guanine nucleotides, suggesting the involvement of GTP-binding proteins. In membranes derived from the liver, however, sodium saccharin had an effect which was dependent on the concentration of membranes used in the adenylate cyclase assay. In high concentrations of membranes, sodium saccharin had a stimulatory effect, while in low concentration an inhibition was observed.

Synthesis of potentially caged sphingolipids, possible precursors of cellular modulators and second messengers.

An increasing number of sphingolipids, glycosphingolipids and some of their degradation products have been recognized in recent years as second messengers involved in signal transduction and as modulators of numerous cellular functions. These can be converted into inert, caged compounds, introduced into cells and tissues and subsequently photolysed to active compounds thus enabling the study of fast biological processes. The novel, potentially caged compounds synthesized here are substituted 2-nitrobenzyl urethans and 2-nitrobenzyl amines derived from sphingosine, dihydrosphingosine, N-methylsphingosine, N-methyldihydrosphingosine, psychosine and glucosylsphingosine. Upon irradiation of the afore mentioned compounds they release, or are expected to release, the free biologically active amines.

Chemoenzymic synthesis of potentially caged glycosphingolipids [correction of glycoshingolipids] (GSLs): potentially caged lyso-G(M3) and its analogue.

In a previous work, a number of potentially caged sphingolipids and glycosphingolipids were chemically synthesized (Zehavi, 1997. Chem. Phys. Lipids 90, 55-61). The effects of GM3 and to a lesser extent, of lyso-GM3, are being studied. Considering that biologically inert, caged lyso-GM3 could be photolysed in the cell to release lyso-GM3, thus creating an attractive opportunity to study the subsequent sequence of events in the cell, the chemoenzymic synthesis of the potentially caged lyso-GM3, (5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylo nic acid)-(2-3)-beta-D-galactopyranosyl-(1-4)-beta-D-glucopyranosyloxy (1-1)- (2S,3R,4E)-2-(4-carboxymethyl-2-nitrobenzyloxycarbonyl-amino)-3-hy droxy-4- octadecene and of a potentially caged GM3 analogue, (5-acetamido-3,5-dideoxy-D- glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-3)-beta-D-galactopyranosyl-(1-4)-beta-D-glucopyranosyloxy -(1-3)- (2S, 3R, 4E)-2-(4-carboxymethyl-2-nitro-benzyloxycarbonylamino)-1- hydroxy-4-octadecene was undertaken. Both compounds, being 2-nitrobenzyloxycarbonyl derivatives, are light-sensitive and could be efficiently photolysed to the biologically active, corresponding lyso-GSLs.

Taste responses to neohesperidin dihydrochalcone in rats and baboon monkeys.

Preference-aversion behavior to solutions containing neohesperidin dihydrochalcone (NHDHC) was studied rats and baboon monkeys. Electrophysiological responses evoked by application of NHDHC solutions to taste receptors innervated by the chorda tympani and the glossopharyngeal nerves were also measured. As a group, rats were indifferent to solutions containing up to 1.2 x 10(-3) M NHDHC in short and long-term preference tests. A solution containing the very high concentration of 8.2 x 10(-3) M NHDHC was consumed less than water by all rats. The aversive behavior of rats to the 8.2 x 10(-3) M NHDHC solution appeared to be due to taste quality rather than olfaction. When percent preferences were calculated on an individual basis for the long-term preference tests, 59% of the rats were indifferent to solutions containing up to 1.2 x 10(-3) M NHDHC, 33% of the animals found this solution aversive and less than 8% showed preference. Behavioral responses to a solution of 3.4 x 10(-4) M aspartame also varied considerably among rats. The electrophysiological data were in line with the behavioral responses suggesting weak taste responses for NHDHC in rats. More pronounced responses observed in the glossopharyngeal nerve as compared to the chorda tympani. Baboon monkeys showed a strong preference for solutions containing 1.6 x 10(-5) M-1.6 x 10(-3) M NHDHC. A solution of 1.6 x 10(-2) M was consumed to a lesser extent than water. It is concluded that baboon monkeys present a better experimental model than rats for investigating the sweetness of NHDHC.

Probing acceptor specificity in the glycogen synthase reaction with polymer-bound oligosaccharides.

Polymers having maltose and maltotriose side-chains were synthesized by attaching 4-carboxy-2-nitrobenzyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside or 4-carboxy-2-nitrobenzyl O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-O-beta-D- glucopyranoside, respectively, to aminoethyl-substituted polyacrylamide gel beads. Subsequently, the two polymers, and analogous polymers having D-glucose and cellobiose side-chains, served in a comparative study as acceptors in the glycogen synthase (UDP-D-glucose: glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) reaction. Highest transfer (4.2%) was observed for the polymer bearing maltotriose groups. The bound saccharides were then removed by irradiation (greater than 320 nm), and examination of them demonstrated that alpha-D-glucosyl oligomerization in the glycogen synthase reaction had occurred.

Polymers having (1----4)- and (1----6)-linked alpha-D-glucopyranosyl groups as acceptors in the glycogen synthase reaction.

Insoluble, light-sensitive polymers linked to maltose, maltotriose, a glycogen-branch point trisaccharide, and panose were synthesized and served in a comparative study as acceptors in the glycogen synthase (UDP-D-glucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11) reaction. The highest transfer rate was observed with the maltotrio polymer. Extending the acceptor linearly with (1----4)-linked alpha-D-glucopyranosyl residues improved the transfer, whereas (1----6)-linked alpha-D-glucopyranosyl branches decreased it.

Synthesis and antifungal activity of medicagenic acid saponins on plant pathogens: modification of the saccharide moiety and the 23 alpha substitution.

The study of structure-antifungal activity relationships of medicagenic acid saponins was widened to include synthetic glycosides of mannose, galactose, cellobiose, and lactose as well as a 23 alpha-hydroxymethyl analog of medicagenic acid, namely, methyl 2 beta,3 beta-dihydroxy-23 alpha-hydroxymethyl-delta (12)-oleanene-28 beta-carboxylate, against Sclerotium rolfsii, Rhizoctonia solani, Trichoderma viride, Aspergillus niger, and Fusarium oxysporum. The native glucose-containing saponin was a more effective antifungal agent than the aforementioned saponins, except in the case of the cellobiose-containing derivative and F. oxysporum. A carboxyl substituent at the 23 alpha position of the sapogenin brought about higher fungistatic activity than a methyl carboxylate which, in turn, was more effective than an hydroxymethyl group at the same position.

Effects of L-cysteine and N-acetyl-L-cysteine on 4-hydroxy-2, 5-dimethyl-3(2H)-furanone (furaneol), 5-(hydroxymethyl)furfural, and 5-methylfurfural formation and browning in buffer solutions containing either rhamnose or glucose and arginine.

Solutions of L-cysteine (Cys) and N-acetyl-L-cysteine (AcCys), containing glucose or rhamnose, with or without arginine, were buffered to pH 3, 5, and 7 and incubated at 70 degrees C for 48 h. Cys and AcCys inhibited the formation of (hydroxymethyl)furfural (HMF) from glucose and methylfurfural (MF) from rhamnose under acidic conditions. AcCys inhibited the accumulation of 4-hydroxy-2, 5-dimethyl- 3(2H)-furanone (DMHF, Furaneol) from rhamnose, but Cys, under our experimental conditions, enhanced Furaneol accumulation from rhamnose. Cys and AcCys reacted directly with Furaneol but not with HMF or MF. Both Cys and AcCys inhibited nonenzymatic browning at pH 7. At pH 3, however, Cys reacted with both glucose and rhamnose to produce unidentified compounds that increased the visible absorbency.

Saponins as antimycotic agents: glycosides of medicagenic acid.

The continuous search for new antimycotic drugs is a consequence of the broad use of immunosuppressive drugs and broad-spectrum antibiotics, high number of AIDS patients, and widespread dermatophyte infections. The concern with increased resistance due to widespread and prolonged antifungal treatment, particularly with azoles, is noteworthy. Our efforts were focused on medicagenic acid derivatives isolated from alfalfa and on semisynthetic ones. In general, these materials exhibited potent fungistatic effects against several plant pathogens and human dermatophytes. Furthermore, they were fungicidal against medically important yeasts, showing a most impressive activity against Cryptococcus neoformans, the minimal fungicidal concentration (MFC) value of the gluco derivative of medicagenic acid, compound G2, is 4 micrograms/ml. The mode of action as well as the structure-activity relationships of these compounds were studied. Compound G2, when applied topically, was effective in curing skin lesions of guinea pigs infected with the dermatophyte Trichophyton mentagrophytes and good skin tolerance to the drug was noted. Furthermore, it had a life-prolonging effect on mice infected with C. neoformans and recently, liposomes containing compound G2 were used efficiently as a drug delivery system in treatment of murine cryptococcosis and candidiosis.

Solid-phase extraction and HPLC determination of 4-vinyl guaiacol and its precursor, ferulic acid, in orange juice.

This study is undertaken to develop a simplified, rapid method to determine both immediate and potential off odors due to 4-vinyl guaiacol and its odorless precursor, ferulic acid, from a single sample preparation and chromatographic analysis. Orange juice sample preparation consists of a simple, C18 solid phase extraction. Utilizing a 5-microns, 25-cm, C18 column, both compounds can be separated within 40 min using a one-step, linear gradient beginning with an aqueous 12% tetrahydrofuran (THF)-5% acetonitrile mixture and ending with 35% aqueous THF. Hesperidin and nariutin have been identified as the compounds that interfered with the ultraviolet (UV) determination of sinapic and caffeic acids. Fluorescence detection with wavelength programming offers optimal sensitivity and selectivity. Recoveries of 4-vinyl guaiacol and ferulic acid range from 90 to 103%. Detection limits are 1 ppm and 5 ppm for ferulic acid and 4-vinyl guaiacol, respectively. Other hydroxycinnamic acids such as coumaric, sinapic, and caffeic acids may also be determined from the same chromatogram.