Asexual reproduction: 'midwives' assist dividing amoebae.
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Animals↗
Biomedical subjects
D Mirelman
Publications and source records attributed to D Mirelman.
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.
Animals↗
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.
Alcohol Dehydrogenase↗
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.
Animals↗
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.
3' Untranslated Regions↗
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.
Cell Membrane Permeability↗
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.
Amebiasis↗
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.
Antineoplastic Agents, Phytogenic↗
Applying antisense technology to the study of entamoeba histolytica pathogenesis: response
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Journal Article↗
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.
Alcohol Dehydrogenase↗
Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence.
One of the under-represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM-1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate-binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.
Animals↗
Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence.
Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 5' and 3' untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by approximately 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.
Animals↗
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.
Animals↗
Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.
Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.
Animals↗
Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal/GalNAc lectin.
Entamoeba histolytica virulence is related to a number of amebic components (lectins, cysteine proteinases, and amebapore) and host factors, such as intestinal bacterial flora. Trophozoites are selective in their interactions with bacteria, and the parasite recognition of glycoconjugates plays an important role in amebic virulence. Long-term monoxenic cultivation of pathogenic E. histolytica trophozoites, strains HK-9 or HM-1:IMSS, with Escherichia coli serotype O55, which binds strongly to the Gal/GalNAc amebic lectin, markedly reduced the trophozoites' adherence and cytopathic activity on cell monolayers of baby hamster kidney (BHK) cells. Specific probes prepared from E. histolytica lectin genes as well as antibodies directed against the light (35-kDa) and heavy (170-kDa) subunits of the Gal/GalNAc lectin revealed a decrease in the transcription and expression of the light subunit in trophozoites grown monoxenically with E. coli O55. This effect was not observed when E. histolytica was grown with E. coli 346, a mannose-binding type I pilated bacteria. Our results suggest that the light subunit of the amebic lectin is involved in the modulation of parasite adherence and cytopathic activity.
Animals↗
Analysis by enzyme-linked immunosorbent assay and PCR of human liver abscess aspirates from patients in China for Entamoeba histolytica.
Aspirates of liver abscesses were analyzed for Entamoeba histolytica. PCR detected a gene encoding a 30-kDa protein in all samples but detected the ribosomal DNA gene in only 14 (33.3%) samples. Enzyme-linked immunosorbent assay detected antigen in 41 (97.6%) samples. PCR analysis of a strain-specific antigen (SSG) revealed that abscesses were caused by various strains.
Animals↗
A spectrophotometric assay for allicin and alliinase (Alliin lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.
Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure alliinase activity and for the activity of the enzyme in crude garlic extracts.
Carbon-Sulfur Lyases↗
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.
Alcohol Dehydrogenase↗