Portable device for magnetic stimulation: assessment survival and proliferation in human lymphocytes.

A device's instrumentation for magnetic stimulation on human lymphocytes is presented. This is a new procedure to stimulate growing cells with ferrofluid in vortices of magnetic field. The stimulation of magnetic vortices was provided at five different frequencies, from 100 to 2500 Hz and intensities from 1.13 to 4.13 mT. To improve the stimulation effects, a paramagnetic ferrofluid was added on the cell culture medium. The results suggest that the frequency changes and the magnetic field variation produce an important increase in the number of proliferating cells as well as in the cellular viability. This new magnetic stimulation modality could trigger an intracellular mechanism to induce cell proliferation and cellular survival only on mitogen stimulated cells.

The effect of biomaterials and antifungals on biofilm formation by Candida species: a review.

Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis are able to form biofilms on virtually any biomaterial implanted in a human host. Biofilms are a primary cause of mortality in immunocompromised and hospitalized patients, as they cause recurrent and invasive candidiasis, which is difficult to eradicate. This is due to the fact that the biofilm cells show high resistance to antifungal treatments and the host defense mechanisms, and exhibit an excellent ability to adhere to biomaterials. Elucidation of the mechanisms of antifungal resistance in Candida biofilms is of unquestionable importance; therefore, this review analyzes both the chemical composition of biomaterials used to fabricate the medical devices, as well as the Candida genes and proteins that confer drug resistance.

Magnetic field influence on electrical properties of human blood measured by impedance spectroscopy.

The impedance spectroscopy technique (IST) was used for studying the effect of a 0.5 T magnetic field on the electrical properties of whole human blood. A Solartron SI 1260 spectrometer was used to measure the impedance spectra of magnetic field exposed blood samples compared to non-exposed samples. An equivalent electrical circuit model, consisting in a resistance Rs in series with a parallel circuit formed by a constant phase element (CPE) and another resistance Rp, is proposed to fit the data in both cases. The experiment used 3 ml human blood samples from 160 healthy donors. A Wilcoxon matched pairs statistical test was applied to the data. The data analysis seems to show a statistically significant increase of the values of resistance Rp (Z = 5.06, P < 0.001) and capacitance CT (Z = 3.32, P < 0.001) of the blood exposed to magnetic field, by approximately 10.4% and 1.9%, respectively.

Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases.

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.

Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica.

Dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the K(m) for the substrate but rather with a 2.6-fold increase in V(max). Phosphorylation in the presence of [gamma-(32)P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of dolichol phosphate mannose synthesis in E.histolytica remains to be determined.

Identification and characterisation of early reactions of asparagine-linked oligosaccharide assembly in Entamoeba histolytica.

Sequential incubation of a mixed membrane fraction isolated from Entamoeba histolytica trophozoites with the nonionic detergents Brij 35 and Igepal CA-630 rendered a soluble fraction with the ability to transfer N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to dolichol phosphate to form a lipid saccharide that was identified as a mixture of dolichol-P-P-GlcNAc and dolichol-P-P-(GlcNAc)2 as follows. (a) The reaction occurred only in the presence of exogenously added dolichol phosphate and was strongly inhibited by tunicamycin and amphomycin; (b) Over 90% of the aminosugar moiety of the lipid saccharide was released by mild acid hydrolysis and was identified as a mixture of GlcNAc and diacetylchitobiose [(GlcNAc)2]; (c) Time course experiments revealed that dolichol-P-P-(GlcNAc)2 accumulated at the expense of a parallel decrease in dolichol-P-P-GlcNAc revealing the tandem operation of UDPGlcNAc:dolichol-P GlcNAc-1-P transferase and UDPGlcNAc:dolichol-P GlcNAc transferase. Mg2+ and to a lower extent Mn2+ were required for catalytic activity and were optimal at 2.5 mM and 1.25 mM, respectively. Common phospholipids with different head groups failed to increase catalytic activity and phosphatidylglycerol was inhibitory. At low concentration, nucleotides such as ATP, GMP and GTP brought about stimulations of 24-54% but higher concentrations were inhibitory. Others were inhibitory at all concentrations the strongest being those containing a uridine base.

Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.

Sequential treatment of trophozoite membranes with the nonionic detergents Brij 35 and Igepal CA-630 released a soluble fraction that efficiently catalyzed the transfer of mannose from GDP-Man into a mannolipid that was identified as dolichol phosphate mannose (Dol-P-Man) by several criteria. The transfer reaction occurred only in the presence of exogenously added dolichol monophosphate (Dol-P). Plots of enzyme velocity versus Dol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values of S0.5 for Dol-P and K(m) for GDP-Man were 15 micrograms/ml and 4.1 microM, respectively. The solubilized fraction failed to transfer the label into other products such as lipid-linked oligosaccharides and glycoproteins. The optimum pH was 7.5-8.0 in potassium phosphate or Tris/HCl buffers and the enzyme required either Mg2+ or Mn2+. The latter was more effective but in a narrower range of concentrations. The transferase was inhibited by a number of nucleotides the strongest being GMP, GDP, and GTP. When assayed in the reverse direction, however, the enzyme catalyzed the transfer of mannose from Dol-P-Man back into GDP-Man as a function of increasing concentrations of GDP. Mg2+ was a better activator of the reverse reaction than Mn2+, which reached up to 60% at 2 mM GDP. These results suggest that some of the enzyme catalytic properties may change depending on the direction of the transfer reaction.

Identification and partial characterization of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin.

Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B', showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed chitinase A as the major species. Chitinase B' was a minor component of the chitinolytic complex. Whereas some properties were common to the three forms, analysis of other parameters revealed significant catalytic site-related differences. Accordingly, the three chitinases hydrolyzed the fluorogenic substrate 4-methylumbelliferyl chitotriose with typical Michaelian kinetics and Km values of 4.5, 11.8 and 3.8 microM for A, B and B', respectively. Allosamidin strongly inhibited the three enzyme forms with different kinetics. Dixon plots revealed competitive-type inhibition and Ki values of 10.0, 2.3 and 10.8 nM for A, B and B', respectively. Km/Ki ratios indicated 450-, 350- and 5130-fold higher affinity for the inhibitor over the substrate for the A, B and B' forms, respectively. Results are discussed in terms of the possibility that the three chitinase species correspond to different enzyme proteins.

Partial purification and characterization of ornithine decarboxylase from Entamoeba histolytica.

Multiplication of E. histolytica was accompanied by a parallel increase in ornithine decarboxylase (ODC) specific activity up to 72 h of cultivation in TYI-S-33 medium. Thereafter, activity rapidly decayed whereas growth continued for another 24 h before entering into the stationary growth phase. ODC was very unstable. Partial purification (14-fold) of the enzyme was achieved by a three-step procedure involving high-speed centrifugation, gel filtration and adsorption to hydroxylapatite. The partially purified enzyme (Mr 211 kDa) revealed maximum activity at pH 8.5-9.0 and a sigmoidal response to substrate concentration. An S0.5 value of 1.0 mM ornithine was estimated. Although ODC did not exhibit an absolute dependence on pyridoxal phosphate (PLP), addition of PLP increased catalytic activity about 4-fold, with an S0.5 value of 45 microM. Evolution of 14CO2 from ornithine was markedly inhibited by polyamines in the following increasing order of effectiveness: putrescine > spermidine > spermine. The substrate analogs alpha-methylornithine and alpha-difluoromethylornithine had no effect on enzyme activity and cell growth. In contrast, 1,3-diaminopropane and 2,4-diamino-2-butanone, 2 putrescine analogs, severely inhibited both enzyme activity and amoeba multiplication. Results are discussed in terms of the role of ODC in the amoeba proliferation.

Ornithine decarboxylase activity in Entamoeba invadens.

Growth of E. invadens was paralleled by a concomitant increase in ornithine decarboxylase activity which peaked after 5 days of cultivation in TYI-S-33 medium. Over this period, enzyme activity increased about nine-fold with respect to that present at the start of incubation. Thereafter and coinciding with the onset of the stationary growth phase, enzyme activity started to decline reaching trace levels after 8 days of cultivation. Most of the enzyme remained soluble following centrifugation of amoeba homogenates at 105,000 g. alpha-Difluoromethylornithine failed to affect ornithine decarboxylase activity in vitro and amoeba growth. The enzyme was markedly inhibited by polyamines (putrescine, spermidine and spermine) and 1,4-diamino-2-butanone, a putrescine-analog. The latter arrested proliferation of cells, an effect that could not be reversed by polyamines which by themselves also inhibited growth to a low but significant extent. Our results indicate that polyamine biosynthesis from ornithine is required for growth of E. invadens and that this function is rapidly abolished following entry into the stationary growth phase.

Encystation in Entamoeba invadens.

Knowledge of the biology and pathogenesis of the human parasite Entamoeba histolytica has been limited by the lack of efficient procedures to induce axenic encystation in the laboratory. However, such methods have been developed for E. invadens, a reptilian parasite, for which encystation can be induced by mechanisms that are as yet poorly defined. This has allowed the analysis of some morphological, physiological and biochemical events that accompany differentiation into cysts. Elucidation of these changes will lead to a better understanding of the process and therefore to the possibility of controlling it. Here, Everardo López-Romero and Julio Cézar Villagómez-Castro emphasize the metabolism of cyst-wall polymers as a potential target to inhibit cyst formation with specific drugs that would be in principle, harmless to the host.

Chitinase activity in encysting Entamoeba invadens and its inhibition by allosamidin.

Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.

Biochemical analysis of Entamoeba histolytica HM1 strain resistant to complement lysis.

In this study, quantitation of several hydrolases and analysis of electrophoretic patterns of E. histolytica HM1 axenic strain periodically exposed to lytic-human serum (lytic-HS), heat-inactivated human (HI-HS) or bovine (HI-BS) sera were performed. The N-acetylglucosaminidase (NAGAse) levels increased in amebas resistant to complement. The acid phosphatase (AP) and proteolytic activity (PA) levels did not vary significantly in all groups of treated amebas, as compared with amebas from axenic cultures. Moreover, amebas exposed to either HI-HS or lytic-HS sera showed a discrete quantitative increase as compared with HI-BS group, in some peptides with a M(r) between 116 and 45 kDa. These results indicate that, besides resistance to complement lysis, exposure of E. histolytica to lytic-HS induces an augmentation in NAGAse levels.

Entamoeba histolytica: a simplified method to quantify its cytotoxicity.

A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.