Benito David Mata-Cárdenas

Synthesis and in vitro antiprotozoal activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazone derivatives

Several thiosemicarbazone derivatives of 5-nitrothiophene-2-carboxaldehyde were prepared by the simple process in which N(4)-thiosemicarbazone moiety was replaced by aliphatic, arylic and cyclic amine. Among these thiosemicarbazones compound 11 showed significant antiamoebic activity whereas compound 3 was more active antitrichomonal than the reference drug.

PEHPS medium: An alternative for axenic cultivation of Entamoeba histolytica and E. invadens

Knowledge of Entamoeba histolytica biology in the last 17 years has been acquired largely as a consequence of this parasites axenic cultivation in TPS-1 or TYI-S-33 media. Unfortunately, there are often low yields in these media, due to variability of their main components, Panmede and yeast extract. We describe a medium, PEHPS, of which the main components are extracts of ox liver, and ox and swine pancreas (EHP). 5 strains of E. histolytica and 2 of E. invadens were quickly and easily adapted to PEHPS and serially cultivated for 3 years. Yields progressively rose initially, and then became stable. Depending on the strain, average yields in the last 6 months of this study were 1.3 to 3.1 x 10(5) amoebae/ml for E. histolytica and 5.5 to 5.7 x 10(5) for E. invadens. All the 18 EHP batches tested supported vigorous amoebal growth. PEHPS had 2 additional advantages: (a) it was stable at 4 degrees C or 25 degrees C for 9 months, and at -10 degrees C for at least 2 years, and (b) it supported amoebal growth with inocula as low as one trophozoite/ml. PEHPS avoids the variability shown by TPS-1 and TYI-S-33, and could therefore be a good alternative for axenic amoebal cultivation.

Differential effects of the (+)- and (–)-gossypol enantiomers upon Entamoeba histolytica axenic cultures

Abstract— The in‐vitro anti‐amoebic effects of (±)‐, (+)‐, (–)‐gossypol and emetine were tested against axenic trophozoites from five Entamoeba histolytica strains. The (–)‐isomer was more active than the racemate and the (+)‐isomer. These results indicate that the gossypol anti‐amoebic activity is mainly due to its content of (–)‐gossypol in all strains tested.

Isolation of an Entamoeba histolytica intracellular alkaline phospholipase A2.

Abstract The major hemolytic activity of Entamoeba histolytica is located in a subcellular fraction called P30. Its maximal effect is observed at pH 8.0 and 1 mM Ca2+ and is due to a phospholipase A (PLA). In the present study a membrane-associated phospholipase A2 was purified from P30 to homogeneity. P30 was fractionated with ethyl ether and the insoluble fraction was extracted with 1 M KCl. The KCl-soluble material was diluted ten times with 0.1 M TRIS-HCl (pH 9.5) and passed through a chromatofocusing column with a 9–4 pH gradient. Four peaks with PLA2 activity were obtained. By affinity chromatography, peak II, the one with the highest specific activity, was resolved in three more PLA2 peaks. Peak II.2 had the highest PLA2 specific activity. When analyzed by sodium dodecyl sulfate-polyacrylamide slab-gel electrophoresis under nonreducing conditions, peak II.2 yielded a single band with an apparent molecular mass of 30 kDa. Under reducing conditions the protein dissociated into two 15-kDa monomers. The purified PLA II.2 displayed its activity at the same conditions under which the P30 hemolytic activity was maximal. The isoelectric point of PLA II.2 was 7.0. The purification procedure described above provides sufficient material for determination of the relative importance of the enzyme in the E. histolytica pathogenic mechanisms.

Identification ofEntamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with14C or3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute toE. histolytica cytopathogenicity.

High susceptibility of five axenic Entamoeba histolytica strains to gossypol

The antiamoebic potency of gossypol was tested against 5 axenic Entamoeba histolytica strains, in logarithmic phase growth in PEHPS medium. All of the strains were moderately susceptible to this polyphenolic drug. The 50% inhibitory concentration (IC50) was of the same order of magnitude in all strains: 0.015 microM (for strain HM-1) to 0.067 microM (for HM-38). The difference between the IC50 of HM-1 and the remaining 4 strains (HM-38, HK-9, HM-2 and HM-3) was significant, although it was greater between HM-1 and HM-38 (P less than 0.005) than between HM-1 and the other 3 strains (P less than 0.05), for which the IC50 was 0.03 microM, 0.049 microM and 0.38 microM respectively. Gossypol is more toxic in vitro for amoebae than other drugs widely used clinically, its pharmacological effects and safe dosage in humans are well known because of its antifertilizing effect, and it is accumulated mainly in the liver and colon. Accordingly, this compound could be a good antiamoebic agent if it is as potent in vivo as it is in vitro.

TRICHOMONAS VAGINALIS: IDENTIFICATION OF SOLUBLE AND MEMBRANE-ASSOCIATED PHOSPHOLIPASE A1 AND A2 ACTIVITIES WITH DIRECT AND INDIRECT HEMOLYTIC EFFECTS

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthals inhibitor. The greatest effect was observed with 80 μM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.

TRICHOMONAS VAGINALIS: IDENTIFICATION OF A PHOSPHOLIPASE A–DEPENDENT HEMOLYTIC ACTIVITY IN A VESICULAR SUBCELLULAR FRACTION

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (S30) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3, GT-13, and GT-15), lysed both human and Sprague–Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0, in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 (6.71 ± 0.33 hemolytic units [HU]/mg/hr to 11.60 ± 0.24 HU/mg/hr) than at pH 8.0 (3.81 ± 0.30 HU/mg/hr to 5.75 ± 0.65 HU/mg/hr), and it was greater than that on human red blood cells at pH 6.0 (2.67 ± 0.19 HU/mg/hr to 4.08 ± 0.15 HU/mg/hr) or pH 8.0 (2.24 ± 0.09 HU/mg/hr to 2.81 ± 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60–93% at pH 6.0 and 78–93% at pH 8.0) by the effect of 80 μM Rosenthals inhibitor, which also inhibited 27–45% and 29–54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.

Synthesis, crystal structure, and enhancement of the efficacy of metronidazole against Entamoeba histolytica by complexation with palladium(II), platinum(II), or copper(II)

Reaction of trans-[PdCl2(DMSO)(2)]. cis-[PtCl2(DMSO)(2)]. and [Cu(OAc)(2)](H2O)-H-. with metronidazole (mnz) leads to the formation of new complexes, i.e., trans-[PdCl2(mnz)(2)] (1), trans-[PtCl2(mnz)(2)] (2). and trans-[Cu-2(OAc)(4)(mnz)(2)] (3), respectively. Complexes 1-3 crystallize all in the centrosymmetric monoclinic space group P2(1)/c with Z = 8. Unit-cell parameters for these complexes are: 1, a = 7.1328(14) Angstrom, b = 20.699(4) Angstrom, c = 7.1455(14) Angstrom, and beta = 116.17(3)degrees; 2, a = 9.9169(14) Angstrom, b = 21.853(4) Angstrom, c = 6.7218(13) Angstrom, and beta = 110.79(3)degrees: 3. a = 9.1663(18) Angstrom, b = 19.129(4) Angstrom, c = 8.9446(18) Angstrom, and beta = 116.44(3)degrees. The complexes 1 and 2 maintain an ideal square-planar geometry. In complex 3, the H2O molecules of the starting complex are replaced by metronidazole while maintaining a dimeric structure of [Cu(OAc)(2)]. Each Cu ion has an ideal octahedral structure, though distortion occurs in the equatorial position where the acetato ligands are attached. The Cu-Cu separation of 2.6343(8) Angstrom indicates considerable metal-metal interaction. The testing of the antiamoebic activity of these complexes against the protozoan parasite Entamoeba histolytica suggests that compound 1-3 might be endowed with important antiamoebic properties since they showed IC50 values in a muM range better than metronidazole (Table 2). Thus, compound 1 displayed more effective amoebicidal activity than metronidazole (IC50 values of 0.103 muM vs. 1.50 muM resp.).

A multipurpose solid-phase method for protein determination with Coomassie brilliant blue G-250

The multipurpose method for protein determination (MMPD) is based in the Coomassie brilliant blue G-250 binding to immobilized and washed proteins in paper filter disks, and the A600 measurement of the eluted protein-dye complexes. The analysis requires 5-microliters samples, put in 7-mm paper filter disks, which can be stored for up to 2 weeks, without sensible changes in their protein content. The A600 is a logarithmic function of the log of bovine serum albumin quantity, between 2.5 and 250 micrograms with two linear segments used as standard curves: one between 2.5 to 20 micrograms and the other between 20 and 80 micrograms. Results with the MMPD were quantitatively comparable with those obtained with the method of Lowry et al., and those reported by other workers, assaying the following material: (i) bovine serum albumin, in doubly distilled water or in presence of several substances that interfere with the current methods; and (ii) total homogenates and their respective trichloroacetic acid-insoluble extracts, which were obtained from several phenol-rich vegetal specimens and complex animal products. The MMPD is proposed as an alternative for protein determination for its versatility and reliability, in both vegetal or animal products, especially when the analysis with traditional methods is made difficult by the presence of natural or added interfering substances and the sample volume is too small to discard them, or when the material must be stored for relatively long periods of time, prior to its processing.