Absence of toxicity and genotoxicity in an extract of Rubus coriifolius.

Rubus coriifolius Focke is a wild plant from the Rosaceae family. It grows in both Guatemala and Mexico. The polar extract of the aerial parts of this plant has antibacterial, anti-inflammatory, and anti-protozoal activities. These properties may explain the traditional use of this plant. In vivo and in vitro assays were used to assess the genotoxic and toxic effects of an ethanol extract of the aerial parts of R. coriifolius. Three groups of rats were orally administered the R. coriifolius extract diluted in ethanol (5%) at doses of 1.89 mg/kg body weight (low dose), 4.72 mg/kg body weight (medium dose), and 9.44 mg/kg body weight (high dose) for 3 weeks. Genotoxic/cytotoxic effects induced by the R. coriifolius ethanol extract were evaluated in vivo by a micronuclei (MN) test in rat's bone marrow cells and in vitro by MN and sister chromatid exchange (SCE) in human lymphocyte cultures. In vivo genotoxicity analyses revealed that the average number of micronucleated polychromatic erythrocytes and the polychromatic erythrocyte/red blood cell ratio at all doses were not significantly different from those of the negative control. In vitro genotoxicity analyses showed that MN, SCE, and proliferative index frequencies in a human lymphocyte cell culture were not significantly different from those of the negative control. These results demonstrate that the ethanol extract of R. coriifolius aerial parts is not toxic or mutagenic (in vitro and in vivo) and does not affect cell proliferation at the concentrations analyzed.

Comparison of gene expression profiles between pansensitive and multidrug-resistant strains of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.

Total homocysteine levels in healthy children from the Monterrey metropolitan area, Mexico.

Currently, there are indications for determining hyperhomocysteinemia in adulthood as risk factors for cardiovascular diseases, psychiatric disorders, pregnancy complications, birth defects, cognitive impairment in the elderly, in addition to cancer. If hyperhomocysteinemia is determined from childhood, it may be modulated with the provision of an opportunity for public health intervention. The objective of this descriptive study was to determine total homocysteine (tHcy) levels in healthy children from the Monterrey metropolitan area in Mexico. In a peripheral-blood sample collected from 56 healthy children aged 2-10 years, we determined tHcy concentration by high performance liquid chromatography (HPLC) with fluorescence detection. The geometric mean +/- SD was 9.78 +/- 1.73 micromol/l. tHcys of the children studied were homogeneous by age cohort and gender. Nutritional state was classified by body mass index (BMI). Sixty five percent of children who participated in the study had normal BMI, and 96% of the children belong to the low socioeconomic status. In conclusion, to our knowledge this is the first-ever information on homocysteine (Hcy) prevalence in a population of healthy Mexican children. tHcy concentration was higher than that reported in other populations studies. This preliminary study could constitute the baseline for future public health studies.

Evaluation of the flora of northern Mexico for in vitro antimicrobial and antituberculosis activity.

The aim of the present study was to evaluate the potential antimicrobial activity of 14 plants used in northeast México for the treatment of respiratory diseases, against drug-sensitive and drug-resistant strains of Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae type b and Mycobacterium tuberculosis. Forty-eight organic and aqueous extracts were tested against these bacterial strains using a broth microdilution test. No aqueous extracts showed antimicrobial activity, whereas most of the organic extracts presented antimicrobial activity against at least one of the drug-resistant microorganisms tested. Methanol-based extracts from the roots and leaves of Leucophyllum frutescens and ethyl ether extract from the roots of Chrysanctinia mexicana showed the greatest antimicrobial activity against the drug-resistant strain of Mycobacterium tuberculosis; the minimal inhibitory concentration (MIC) were 62.5, 125 and 62.5 microg/mL, respectively; methanol-based extract from the leaves of Cordia boissieri showed the best antimicrobial activity against the drug-resistant strain of Staphylococcus aureus (MIC 250 microg/mL); the hexane-based extract from the fruits of Schinus molle showed considerable antimicrobial activity against the drug-resistant strain of Streptococcus pneumoniae (MIC 62.5 microg/mL). This study supports that selecting plants by ethnobotanical criteria enhances the possibility of finding species with activity against resistant microorganisms.

A modified chemical protocol of decellularization of rat sciatic nerve and its recellularization with mesenchymal differentiated schwann-like cells: morphological and functional assessments.

The functional reconstruction of large neural defects usually requires the use of peripheral nerve autografts, though these have certain limitations. As a result, interest in new alternatives for autograft development has risen. The acellular peripheral nerve graft is an alternative for peripheral nerve injury repair, but to date there is not a standardized chemical decellularization method widely accepted. The objective of this study was to propose a modified chemical protocol of decellularization of rat sciatic nerve and its recellularization in vitro with mesenchimal differentiated Schwann-like cells. After the transplantation, an evaluation of its regeneration was performed using morphological and functional tests. The study consisted of two phases; in phase 1, different concentrations and times of exposure of rat sciatic nerves to detergents were tested, to establish a modified chemical protocol for nerve decellularization. The chemical treatment with 3% triton X-100 and 4% sodium deoxycholate for 15 days allowed a complete decellularization whilst conserving the extracellular matrix of the harvested nerve. In phase 2, the decellularized and recellularized alografts were compared against autografts. The morphological analysis showed a higher positivity to specific myelin antibodies in the recellularized group compared to the autograft. There were no differences in this parameter between the control limb and the experimental limb (recellularized group). The functional analysis showed no statistical differences at week 15 in the Sciatic Function Index in the autograft group vs the other groups. This study sets the morphological and functional bases for posterior studies about nerve defects regeneration in humans.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans.

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Characterization of chitin synthases from Entamoeba.

A major component of the Entamoeba cyst wall is chitin, a homopolymer of beta-(1,4)-linked N-acetyl-D-glucosamine. Polymerization of chitin requires the presence of active chitin synthases (CHS), a group of enzymes belonging to the family of beta-glycosyl transferases. CHS have been described for fungi, insects, and nematodes; however, information is lacking about the structure and expression of this class of enzymes in protozoons such as Entamoeba. In this study, the primary structures of two putative E. histolytica CHS (EhCHS-1 and EhCHS-2) were determined by gene cloning and homologous proteins were identified in databases from E. dispar and the reptilian parasite E. invadens. The latter constitutes the widely used model organism for the study of Entamoeba cyst development. The two ameba enzymes revealed between 23% and 33% sequence similarity to CHS from other organisms with full conservation of all residues critically important for CHS activity. Interestingly, EhCHS-1 and EhCHS-2 differed substantially in their predicted molecular weights (73 kD vs. 114 kD) as well as in their isoelectric points (5.04 vs. 8.05), and homology was restricted to a central stretch of about 400 amino acid residues containing the catalytic domain. Outside the catalytic domain, EhCHS-1 was predicted to have seven transmembrane helices (TMH) of which the majority is located within the C-terminal part, resembling the situation found in yeast; whereas, EhCHS-2 is structurally related to nematode or insect chitin synthases, as it contained 17 predicted TMHs of which the majority is located within the N-terminal part of the molecule. Northern blot analysis revealed that genes corresponding to CHS-1 and CHS-2 are not expressed in Entamoeba trophozoites, but substantial amounts of CHS-1 and CHS-2 RNA were present 4 to 8 hours after induction of cyst formation by glucose deprivation of E. invadens. The time-courses of expression differed slightly between the two ameba CHS genes, as in contrast to CHS-1 RNA, expression of CHS-2 RNA was more transient and no plateau was observed between 8 and 16 hours of encystation. However, both CHS RNAs were no longer detectable after 48 hours when most of the cells had been transformed into mature cysts.

Cytopathogenicity of Entamoeba histolytica: hemolytic activity of trophozoite homogenates.

In in vitro assays the hemolytic activity of total homogenates of trophozoites of seven strains of amebae were tested for specificity and potency against the erythrocytes of eight mammalian species. The species of Entamoeba included in the tests were E. histolytica, E. invadens, E. moshkovskii, and the Laredo type of E. histolytica. The hemolytic activity was found to be dose-dependent and strain-specific, with E. histolytica being generally the species having the highest hemolytic potency.

In vitro anti-trichomonad effectiveness of a gossypol-metronidazol blend.

With the aid of the Basic Sequential Analytical Simplex Method the in vitro anti-trichomonad effectiveness of several metronidazole/gossypol blends was determined. While the combination that produced 50% of growth inhibition on trichomonad cultures (IC50) contained 15.1 microM gossypol and 0.087 microM metronidazole, the IC(50) of metronidazole and gossypol tested separately was 38.08 microM and 0.195 microM, respectively. Thus, the IC50 of mixed gossypol and metronidazole was, respectively 60.34 and 55.4% lower than that of each separated drug. Whereas metronidazole is usually administered orally, gossypol, used as spermicide, is formulated as pessary. In such conditions the absorption of metronidazole is very efficient while that of gossypol is strikingly poor. The above suggest that a gossypol-metronidazole anti-trichomoniasis therapeutic scheme could be as effective as metronidazole alone, but less toxic.

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.

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 parasite's 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.

Immediate cell membrane damage produced by Entamoeba histolytica subcellular extracts.

The alpha[14C]amino isobutyric acid is a synthetic amino acid with low molecular weight, not incorporated to protein. Chinese hamster ovary (CHO) cells were labeled with this radioactive compound and treated with the amebal subcellular fraction P30 at short times. We detected membrane damage in CHO cells by specific radiolabel release from the first 30 sec of interaction between cell cultures and amebal fraction.

Comparable growth of a Trichomonas vaginalis strain in PEHPS and TYI-S-33 media.

PEHPS medium, developed for axenic cultivation of Entamoeba histolytica and E. invadens, was also capable of supporting the growth of a Trichomonas vaginalis strain, with an inoculum of 1 to 100 trichomonads/ml. The logarithmic growth phase in PEHPS or in TYI-S-33 medium lasted 72 h; yield (3.33 +/- 0.56 x 10(6) trichomonads/ml), duplication time (4.27 h), number of duplications (16.85), or increase ratio (33,328) in PEHPS medium showed no significant differences with those obtained in TYI-S-33 under similar culture conditions. Accordingly, PEHPS medium might be used for the axenic cultivation of T. vaginalis.

Entamoeba histolytica: inhibition of malic enzyme and alcohol dehydrogenase by (+/- )-, (+)-, and (-)- gossypol.

Gossypol, a natural racemic mixture with action on NADP- and NAD-oxidoreductases from diverse species, has been proposed as a possible antiamebic medication considering several of its pharmacological properties. In this study it was found that malic enzyme and alcohol dehydrogenase from Entamoeba histolytica are strongly inhibited by (+/-)-gossypol, and both (+)- and (-)- enantiomers. The inhibition was of the noncompetitive type among their respective substrates in all cases. The (+/-), (+)-, (-)-gossypol half-maximal inhibitory concentrations (IC50) for the malic enzyme were 3.71, 13.37 and 1.03 microM, and against the alcohol dehydrogenase 79.64, 124.43 and 42.56 microM, respectively. Therefore, the (-) enantiomer resulted 3.6 and 13.0 times more potent than the racemic mixture and (+)-gossypol, respectively, to inhibit the malic enzyme, and 1.9 times and 2.9 times more potent than the racemic mixture and (+)-gossypol, respectively, against the alcohol dehydrogenase. Accordingly, one possible mechanism of the antiamebic effect of gossypol could be the inhibition of vital NADP-dependent enzymes as those analyzed in this study.

Entamoeba histolytica axenic growth improvement by ox bile.

Axenic Entamoeba histolytica cultures, grown in PEHPS medium, showed increasing yields and growth velocity when added with 0.03 to 0.6 g/l gallbladder ox bile, while higher doses were toxic for amebas. The highest density (505 +/- 29 x 10(3) trophozoites/ml) and shortest duplication time (Dt = 10.74 +/- 0.2 h) corresponded to cultures added with 0.24 g bile/l. Their yields were 1.74 and 3.34 times higher, respectively, than those which were reached by the non-added PEHPS controls and by growth of amebas in TYI-S-33 medium. Between 72 and 96 h of incubation a noticeable increase in trophozoite density was observed in cultures added with 0.24 g/1 bile among controls. At 72 h yields of bile-added cultures inoculated with 5, 10 and 20 x 10(3) amebas/ml increased in function of the inoculum. The improved growth of E. histolytica by adding 0.24 g/l ox bile to culture medium suggests that bile has compounds are essential for amebas.

Further studies on the in vitro activity of gossypol as antiamebic agent.

Gossypol has an in vitro antiamebic effect, which is 11,39, and 980 times more potent than emetime, metronidazole and diiodohydroxyquinolein, respectively, on five Entamoeba histolytica axenic strains. The trophozoite NADP-dependent malic enzyme and alcohol dehydrogenase are inhibited by (+/-)-gossypol, in a noncompetitive manner with respect to the substrate. From the (-)- and (+)- enantiomers, which integrate the natural gossypol mixture, the first one is three and four times more active in inhibiting both the amebic culture growth and the mentioned oxidoreductases, respectively. The above results justify the analysis of gossypol as an antiamebic drug in experimentally infected animals.

Mg, Mn, and Co ions enhance the formation of Entamoeba histolytica cyst-like structures resistant to sodium dodecyl sulfate.

BACKGROUND: Entamoeba histolytica forms cyst-like structures (CLS) in PEHPS but not in TYS-33 medium. Sodium dodecyl sulfate [(SDS (0.1%)] dissolves most of them in 10 min, but not natural cysts. Chitin is responsible mainly for cyst wall resistance. Its synthesis depends on Mg(2)+, Mn(2)+, or Co(2)+, whose action is interactive. With the aid of the Simplex method, we analyzed the effect of 20 blends of these cations to find the one that, when added to PEHPS, produced the highest proportion of CLS resistant to 1% SDS (RCLS). METHODS: The concentration of Mg(2)+, Mn(2)+, and Co(2)+ was determined in PEHPS and TYI-S-33 with a flame atomic absorption spectrometer. The proportion of RCLS produced in PEHPS with each ion blend was tested. The CLS and RCLS affinity to fluorescein wheat germ agglutinin (WGA/FITC), which binds chitin, was determined. RESULTS: PEHPS contained a similar concentration of Co(2)+ (0.52 microM) and 3.4 and 1.6 times more Mg(2)+ (798 microM) and Mn(2)+ (3.15 microM) than TYI-S-33, respectively. The proportion of RCLS increased gradually in PEHPS until reaching 3.6 +/- 1.43% with MgCl(2) 1.22 mM, MnCl(2) 14.44 mM, and CoCl(2) 19.44 mM (ion blend No. 20). Both CLS and RCLS bound WGA/FITC. The RCLS formed in the presence of ion blend No. 20 appeared wrinkled. CONCLUSIONS: Mg(2)+, Mn(2)+, and Co(2)+ enhanced the ability of PEHPS to form RCLS, possibly because these ions stimulated their chitin synthesis. Although ion blend No. 20 produced the highest proportion of RCLS, this high ion concentration may be toxic for encysting amebas.

Axenic massive cultivation of Entamoeba histolytica trophozoites in spinner flasks and in a microfermentor.

A medium, PEHPS, whose main components are extracts of ox liver, and ox and swine pancreas (EHP) was used to develop two alternative methods for axenic cultivation of trophozoites in suspension: with magnetic stirrer and with a 14 l capacity microfermentor, giving the first technic yields of 2.36 x 10(5) to 3.08 x 10(5) and the second in a 14 l capacity microfermentor, which produced 1.65 x 10(5) amebas/ml in one batch and 2.94 to 3.65 x 10(5) of such parasites/ml at semicontinuous flux.

Detection of a factor released by L5178Y lymphoblasts that inhibits mouse macrophage-activation induced by lipopolysaccharides.

BACKGROUND: Several factors inhibit cellular immune response by deactivating macrophages, but very few, such as those described here, prevent macrophage activation. METHODS: Ascites liquid from 12-day-old BALB/c mice bearing 5178Y lymphoma tumors was collected, and cell-free ascites liquid (CFAL) was separated from lymphoblasts. The supernatant (S1) was obtained from the homogenized and centrifuged lymphoblasts. Then, macrophage cultures containing 0.2 x 10(6) cells from lymphoma-bearing or healthy mice were added to 10 microL of CFAL or S1, plus 5 micrograms of lipopolysaccharides (LPS)/mL, 40 U interferon-gamma or a blend of both. Macrophages were incubated with CFAL or S1 prior to or after adding the activators to investigate whether any of the previously mentioned lymphoma fractions inhibited macrophage activation or whether they deactivated them. The effect of CFAL or S1 was estimated as the diminution of the amount of nitric oxide released by the experimental macrophage cultures with respect to controls (activated macrophages treated with none of the lymphoma fractions). RESULTS: LPS, IFN-gamma, and the LPS/gamma blend activated macrophages from both lymphoma-bearing and healthy mice. None of the lymphoma fractions deactivated macrophages. CFAL, but not S1, inhibited the macrophage activation, i.e., the percentage of inhibition of nitric oxide releasing 76.7% and 78.1% in macrophages from healthy and lymphoma-bearing mice, respectively. In addition, CFAL was unable to inhibit the macrophage-activation effect of IFN-gamma or the LPS/IFN-gamma blend. CONCLUSIONS: Mouse L5178Y lymphoma releases a factor that in vitro inhibits the macrophage activation induced by LPS, but not by IFN-gamma controls.

Orally administered gossypol has no effect on eight hamster erythrocytic enzymes.

The effectiveness of gossypol as an antifertilizing agent is due to the severe injuries or death that this drug produces on spermatozoa and spermatides. Several in vitro and in vivo studies have shown that spermatozoal lactic and malic dehydrogenases are inhibited by gossypol; and that these are more susceptible than the somatic enzymes. Notwithstanding, the in vivo effects on other somatic enzymes have been poorly analyzed. The present study shows that gossypol did not produce toxic effects on eight erythrocytic enzymes of male hamsters that were fed daily with 20 mg of gossypol/kg, for 1, 3, 5 or 10 days. The enzymatic activities analyzed were: adenylate kinase, hexokinase, glucose-6-phosphatase, glucose phosphoisomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglyceratokinase and pyruvate kinase.