S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 microM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 microM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

Engineering of chicken avidin: a progressive series of reduced charge mutants.

Avidin, a positively charged egg-white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly. The high pI of avidin (approximately 10.5), however, is a hindrance in certain applications due to non-specific (charge-related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin-related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical biosensor interaction analysis. In most cases, their thermal stability characteristics were indistinguishable from those of the wild-type avidin. Our results demonstrate that the charge properties of avidin can be modified without disturbing the crucial biotin-binding activity.

Allicin from garlic strongly inhibits cysteine proteinases and cytopathic effects of Entamoeba histolytica.

The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

The epitopes for natural polyreactive antibodies are rich in proline.

"Natural" polyreactive antibodies, which bind in a nonspecific manner to a range of biological molecules both of self- and nonself- origin, are normal constituents of serum and are a significant part of the immune repertoire in many species, including humans. Autoantibodies to sTNF-R (the 55-kDa extracellular domain of the human receptor to tumor necrosis factor alpha) were affinity purified from normal human sera using immobilized sTNF-R. The isolated anti-sTNF-R IgG bound both native and denatured forms of the receptor with low affinity. These antibodies also bound to different proteins and therefore are considered to be polyreactive. We used the anti-sTNF-R antibodies and purified polyreactive antibodies to mannose-specific lectin from garlic (Allium sativum) for screening a peptide library displayed on filamentous M13 phage. After the biopanning procedure, we failed to find epitopes with a consensus sequence; however, we found that proline is the most frequent amino acid in the selected phagotopes. Proline is commonly present at solvent-exposed sites in proteins, such as loops, turns, N-terminal first turn of helix, and random coils. Thus, structures containing proline can serve as conformation-dependent common "public" epitopes for polyreactive natural antibodies. Our findings may be important for understanding polyreactivity in general and for the significance of polyreactive natural antibodies in immunological homeostasis.

Salicylaldehyde-metal-amino acid ternary complex: a new tool for immobilized metal affinity chromatography.

An immobilized salicylaldehyde (sal) was used to build various salicylaldehyde-copper-amino acid (Sal-Cu-AA) complexes which are stable at a range of pH values (2.0-11.0). The complexes were found to bind protein molecules as IMAC resins. Thirteen proteins were examined for their binding to a Sal-Cu-Gly column. The efficacy of the Sal-Cu-AA resin for protein separation were demonstrated by two examples. The first was a new purification process for garlic lectins from garlic crude extract. It seems that in this case the Sal-Cu-AA resins were more selective than IDA resin. The second was immobilization of concanavalin A (Con A) on the resin and using the immobilized Con A for affinity chromatography of mannose-rich glycoprotein ovalbumin. The Con A could be later eluted with EDTA or imidazole and the Sal-containing polymer could be recharged again for further use.

Alliinase (alliin lyase) from garlic (Alliium sativum) is glycosylated at ASN146 and forms a complex with a garlic mannose-specific lectin.

Alliinase (EC 4.4.1.4) catalyses the production of allicin (thio-2-propene-1-sulfinic acid S-allyl ester), a biologically active compound which is also responsible for the characteristic smell of garlic. It was demonstrated that alliinase which contains 5.5-6% of neutral sugars, gives clear PAS-staining, binds to Con A and can form a complex with garlic mannose-specific lectin (ASA). Evidence that the formation of such a complex is mediated by the interaction of the carbohydrate of the glycoprotein enzyme with the lectin was obtained from a radioligand assay which demonstrated the binding of alliinase to ASA and competitive inhibition of this binding by methyl alpha-D-mannoside. ASA I was shown as the lectin mainly present in the complex with alliinase. The results of this study also demonstrate that alliinase is glycosylated at Asn146 in the sequence Asn146-Met147-Thr148.

Natural antibodies to dietary proteins: the existence of natural antibodies to alliinase (Alliin lyase) and mannose-specific lectin from garlic (Allium sativum) in human serum.

It is known that human serum contains natural antibodies to self and non-self proteins. We wished to determine whether normal human serum contains antibodies to dietary proteins that were never injected. We found that human serum contains antibodies to the two major proteins from cloves of garlic (Allium sativum) which is used as a flavorigard dietary food additive. The antibodies found were directed against alliinase and mannose-specific Allium sativum agglutinin (ASA). The antibodies were purified by affinity chromatography on their corresponding antigens. The purified immunoglobulins were mainly of the IgG and IgM classes and could be divided into two categories--specific and crossreactive. The anti-alliinase antibodies were highly specific, while anti-ASA antibodies were polyreactive. Some of the possible reasons for this difference in specificity are suggested.

Monoclonal anti-biotin antibodies simulate avidin in the recognition of biotin.

The sequence of the VH gene of a monoclonal anti-biotin antibody was determined. Biotin-binding motifs, similar to those in avidin and streptavidin, were identified in complementary determining regions 2 and 3, suggesting that natural selection of functional motifs may occur in unrelated protein types.

Localization of serotonin receptors in the rat thalamus by electrophysiology and the action of 5-HTP-DP-hex.

This study was designed to pinpoint the site at which N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP-hex) exerts its previously reported effect on thalamic neurons in rats. The animals were prepared under chloralose-urethane anesthesia for a stereotaxic approach to either the nucleus ventralis posterolateralis (nVPL) or the centrum medianum-parafascicularis complex (CM-Pf) of the thalamus. Individual neurons in these nuclei were separately activated by single-pulse stimulation of the sciatic nerve or the thalamic fibers that form reciprocal connections between the CM-Pf and the second somatosensory (SII) region of the nVPL. Poststimulus time histograms were constructed from computer readouts of the stimulus-evoked responses of a neuron during a 500-ms period accumulated in a digital computer 100X. In addition, the number of spikes accumulated in each histogram was compared to the number of spikes accumulated under identical conditions on the same neuron after intracarotid infusion of 5-HTP-DP-hex. The effect of the drug was reversed by the infusion of 5-HTP. Statistical evaluation of the accumulated spike counts indicated that 5-HTP-DP-hex suppressed only the excitation of CM-Pf neurons from the SII of the nVPL; the input of the sciatic nerve into the CM-Pf remained unaltered. Furthermore, no effect was exerted by this dipeptide on the afferent excitation of neurons in the SII of the nVPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of covalently bound silica-nitroimidazole drug particles on Entamoeba histolytica.

Most commonly used antiamoebic drugs are effective in invasive amebiasis, but their response against trophozoites of Entamoeba histolytica, present in the lumen of the human colon, is inadequate. We report the development of an antiamoebic drug carrier that may be effective against luminal infections. Our preparation consists of small silica particles (5-10 microns in diameter) covalently linked to a potent antiamoebic drug, 2-(4-aminophenoxymethyl)-5-nitro-1-methyl imidazole. Silica-drug particles were injected into mice, hamsters, and guinea pigs. We found that trophozoites phagocytosed the particles in vivo and in vitro, followed by rapid cell death due to the released drug. Analysis of mouse serum revealed that no drug was absorbed from the intestine after placement of the drug-containing particles in the intestine. The antiamoebic activity of particles recovered from the intestine was almost fully retained. This novel antiamoebic concept may be useful for luminal therapy for asymptomatic amebiasis and may minimize side effects and frequency of administration.

Specific localization and quantification of biotin transport components in yeast by use of a biotin-conjugated, impermeant, electron-dense label.

Two approaches are described for the localization and quantification of biotin transport components in yeast cells. One approach is based on tracing the fate of a radioactive affinity label for the biotin transport system, [14C]biotinyl-p-nitrophenyl ester (pBNP), through various stages of subcellular fractionations. A complementary method involves the use of a biotin-derivatized, impermeant, electron-dense, affinity-cytochemical label (ferritin-biotin conjugates) for subsequent visualization by electron microscopy. Values of approximately 8,000 and 4,000 sites/cell, respectively, were achieved by the two methods. Complicating factors, future perspectives and the relevance of the two methods to the isolation of transport components are discussed.