Human recombinant glutamate oxaloacetate transaminase 1 (GOT1) supplemented with oxaloacetate induces a protective effect after cerebral ischemia.

Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate oxaloacetate transaminase 1 (GOT1) activation by means of oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of oxaloacetate seems to be a successful approach for stroke treatment.

Novel derivatives of 6-mercaptopurine: synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines.

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

Trophozoites of Entamoeba histolytica epigenetically silenced in several genes are virulence-attenuated.

The human intestinal parasite Entamoeba histolytica causes amoebic colitis and amoebic liver abscesses. Three classes of amoebic molecules have been identified as the major virulence factors, the Gal/GalNAc inhibitable lectin that mediates adherence to mammalian cells, the amoebapores which cause the formation of membrane ion channels in the target cells and the cysteine proteinases which degrade the matrix proteins, the intestinal mucus and secretory IgA. Transcriptional silencing of the amoebapore (Ehapa) gene occurred after transfection of trophozoites with a plasmid containing a segment of the 5' upstream region of the gene. Transcriptional silencing of the Ehap-a gene continued even after the removal of the plasmid and the cloned amoebae were termed G3. Transfection of G3 trophozoites with a plasmid construct containing the cysteine proteinase (EhCP-5) gene and the light subunit of the Gal- lectin (Ehlgl1) gene, each under the 5' upstream sequences of the amoebapore gene, caused the simultaneous epigenetic silencing of expression of these two genes. The resulting trophozoites, termed RB-9, were cured from the plasmid and they do not express the three types of virulent genes. The RB9 amoeba are virulence attenuated and are incapable of killing mammalian cells, they can not induce the formation of liver abscesses and they do not cause ulcerations in the cecum of experimental animals. The gene-silenced amoebae express the same surface antigens which are present in virulent strains and following intra peritoneal inoculation of live trophozoites into hamsters they evoked a protective immune response. Further studies are needed to find out if RB-9 trophozoites could be used for vaccination against amoebaisis.

[3H]Allicin: preparation and applications.

Allicin (diallylthiosulfinate), the active substance of garlic, has been shown to possess a variety of biological activities. Mechanistic and pharmacokinetic studies of allicin and its derivatives raise the need for a labeled compound. However, labeling of this volatile and unstable liquid requires delicate handling. Here, we describe a simple method for the preparation of (3)H-labeled allicin. This was achieved by applying synthetic [(3)H]alliin ([2,3-(3)H]allylcysteine sulfoxide) to a column containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [(3)H]allicin was done by differential adsorbtion of the reaction components on a neutral polystyrene resin, Porapak Q. Thiol-containing compounds are known to be the main target of allicin. In this work we demonstrated that [(3)H]allicin can be used for the synthesis of labeled [(3)H]allylmercapto derivatives of SH peptides and proteins. Thus, we prepared [(3)H]S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we showed that incubation of alliinase with [(3)H]allicin led to modification of 1.4 cysteine residues per subunit of the enzyme.

The effects of allicin and enalapril in fructose-induced hyperinsulinemic hyperlipidemic hypertensive rats.

The effects of a synthetic preparation of an active constituent of garlic, allicin, were studied on blood pressure (BP), triglycerides, and insulin levels in Sprague-Dawley rats in which high fructose feeding elicited hyperinsulinemia, hypertension, and hypertriglyceridemia. Results were compared with those of the antihypertensive drug enalapril. Three groups of male Sprague-Dawley rats were fed a fructose-enriched diet for 5 weeks. During the last 2 weeks 10 animals received only fructose, 10 received allicin, and 10 received enalapril. Blood pressure, insulin level, and triglyceride levels were measured at the beginning of the experiment and after 3 and 5 weeks on the fructose diet, fructose/allicin diet, or fructose/enalapril diet. Allicin lowered BP from the maximal level (after 3 weeks of fructose) of 153.4 +/- 8 mm Hg to 139.7 +/- 12 mm Hg after 2 weeks on allicin; insulin from 11.7 +/- 3.7 ng/mL on fructose diet to 6.92 +/- 3.3 ng/mL on allicin; and triglycerides from 132.8 +/- 18 mg/dL on fructose to 59.6 +/- 27 mg/dL on allicin. The similar effect of allicin and enalapril on BP, insulin, and triglycerides reinforces the trend toward combining the nonpharmacologic approach with drug therapy.

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.

Entamoeba histolytica cysteine proteinases with interleukin-1 beta converting enzyme (ICE) activity cause intestinal inflammation and tissue damage in amoebiasis.

The protozoan parasite Entamoeba histolytica causes intestinal inflammation and ulceration. Amoebic trophozoites activate the transcription factor NF-kappa B in human intestinal epithelial cells, initiating an inflammatory response programme with resultant damage to the intestinal tissue. Amoebic cysteine proteinases have been proposed as important virulence factors for amoebiasis. To test the role of amoebic cysteine proteinases in the pathogenesis of amoebic colitis, human intestinal xenografts in SCID mice were infected with E. histolytica trophozoites expressing an antisense message to ehcp5. The cysteine proteinase-deficient amoeba failed to induce intestinal epithelial cell production of the inflammatory cytokines interleukin (IL)-1B and IL-8, and caused significantly less gut inflammation and damage to the intestinal permeability barrier. The critical role of amoebic cysteine proteinases in human gut inflammation and tissue damage may be explained by our discovery that amoebic cysteine proteinases possess IL-1B converting enzyme (ICE) activity. This ICE activity could contribute to intestinal inflammation by activating human pIL-1B released by damaged intestinal cells. These results demonstrate for the first time that amoebic cysteine proteinases are a key virulence factor in amoebic colitis, and provide a novel mechanism for their activity.

Inhibition of gene expression in Entamoeba by the transcription of antisense RNA: effect of 5' and 3' regulatory elements.

Down regulation of gene expression by antisense RNA is one of the ways to investigate the specific contribution of certain components to the physiology and activities of a cell. A successful inhibition of gene expression in Entamoeba trophozoites was achieved in stable transfectants by using hybrid plasmid constructs containing promotors that produce transcripts which do not bind to polysomes. Different promotors were found to be required for Entamoeba histolytica or Entamoeba dispar. In E. histolytica one of the two copies (g34) of the gene coding for ribosomal protein L21 was previously found to be transcribed but not translated. Inhibition of gene expression was obtained by placing in a transfection vector, the amoebapore A gene, in its antisense orientation, under the control of the g34 promotor. Transfectants of E. histolytica were shown to accumulate antisense transcripts and inhibit amoebapore synthesis. In contrast, transfectants with plasmid constructs in which the amoebapore gene was placed under the control of the gLE3 promotor of RP-L21, which is known to be translated, did not accumulate antisense transcript or inhibit gene expression. Maximal inhibition of amoebapore expression was obtained when the antisense construct also included the 5' and 3' untranslated regions of the amoebapore gene. In E. dispar the opposite situation was found, plasmid constructs containing the promotor regions of the gLE3 copy, which were shown to be poorly translated, were more efficient in inhibiting the synthesis of a 30 kDa surface-specific antigen than a construct with the g34 promotor element.

The major surface antigens of Entamoeba histolytica trophozoites are GPI-anchored proteophosphoglycans.

Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcalpha1-6](n)Glcbeta1-6Gal (where n=2-23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal(1)Man(2)GlcN-myo-inositol, and in being heterogeneously modified with chains of alpha-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50-180 kDa (PPG-1) and 35-60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcbeta1-6Gal). However, the PPGs are abundant in all strains (8x10(7) copies per cell) and are likely to form a protective surface coat.

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.

Antimicrobial properties of allicin from garlic.

Allicin, one of the active principles of freshly crushed garlic homogenates, has a variety of antimicrobial activities. Allicin in its pure form was found to exhibit i) antibacterial activity against a wide range of Gram-negative and Gram-positive bacteria, including multidrug-resistant enterotoxicogenic strains of Escherichia coli; ii) antifungal activity, particularly against Candida albicans; iii) antiparasitic activity, including some major human intestinal protozoan parasites such as Entamoeba histolytica and Giardia lamblia; and iv) antiviral activity. The main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essential metabolism of cysteine proteinase activity involved in the virulence of E. histolytica.

Allicin-induced decrease in formation of fatty streaks (atherosclerosis) in mice fed a cholesterol-rich diet.

BACKGROUND: Garlic (Allium sativum) has been considered to exhibit therapeutic features for many years. The effects of garlic on levels of serum lipids and on atherosclerosis have been investigated extensively. We have previously demonstrated that allicin, an active component of garlic, exerts a beneficial effect on lipid profile in hyperlipidemic rabbits. OBJECTIVE: To investigate the effects of allicin on formation of fatty streaks (atherosclerosis) and lipid profile in mice. METHODS: Allicin was extracted from garlic and kept in a buffer citrate solution at 4 degrees C. Sixty C57BL/6 mice were fed Paigen diet (17% fat, 1.25% cholesterol) for 15 weeks. Thirty randomly selected animals were administered allicin solution (9 mg/kg) and 30 were administered placebo. Blood lipid profile was evaluated five times during the study. At the end of the 15-week period, the animals were killed and the aortic sinus was evaluated for formation of fatty streaks (atherosclerosis). RESULTS: We observed no statistically significant differences between blood lipid profiles of groups. Microscopic evaluation of aortic sinus formation of fatty streaks (atherosclerosis), however, showed that values for mice in the allicin-treated group were significantly lower: areas of formation of fatty streaks (atherosclerosis) were 13,440 +/- 3310 and 23,410 +/- 3723 micron 2, respectively, for allicin-treated and control mice (means +/- SEM; P = 0.023). CONCLUSIONS: These results indicate that allicin reduces formation of fatty streaks (atherosclerosis) in hyperlipidemic mice. These changes do not seem to occur through an alteration in blood lipid profile.