Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Photosynthetic electron transport interaction of xanthorrhizol isolated from Iostephane heterophylla and its derivatives

A bioactivity-guided chemical study of Iostephane heterophylla (Asteraceae) led to the isolation of xanthorrhizol (1) as the compound that causes inhibition of ATP synthesis, H⁺-uptake and electron flow from water to methylviologen (basal, phosphorylating and uncoupled) in freshly lysed spinach chloroplasts, thus acting as an inhibitor of the Hill reaction. Acetyl (2), dihydro (3) and acetyl-dihydro (4) derivatives were synthesized. It was found that 4 was less active than 1 and 2 in ATP synthesis, whereas 3 was the most potent inhibitor of the Hill reaction and was also an inhibitor of H⁺-ATPase. Studies of the photosynthetic partial redox reactions from PQ to MV indicated that 1 partially inhibited the PQ pool, but that 3 did not. However, both inhibited the uncoupled electron transport in PSII from water to DCBQ. Uncoupled electron flow from water to silicomolybdate was completely inhibited by 3 and partially by 1. The reaction from DPC to DCPIP was inhibited by both 1 and 3. These results indicate that the inhibition site is located within PSII for 1 and 3 as was corroborated by fluorescence decay data.     

A rapid extraction method for detecting aflatoxin producing isolates

A rapid extraction method for screening aflatoxin producing potential ofAspergillus flavus group isolates is described. The method is performed using a moist wheat medium with ca. five infected grains extracted with 2 mL of chloroform, and using thin layer chromatography. This method was proved with 95A. flavus isolates from animal feeds.     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

Superoxide dismutase in strains of the genus Flavobacterium: isolation and characterization

Two electrophoretically different forms of superoxide dismutase, one of them containing manganese-protein and the other iron-protein, were detected in eleven different strains of the genus Flavobacterium. The activities of the different strains were similar to those described for other bacteria. The two molecular forms of the enzyme differed clearly with regard to activity, electrophoretic behaviour, sensitivity to cyanide and peroxide, and NaCl requirement. Both molecular forms were isolated from Flavobacterium halmephilum. Molecular mass absorption spectra, metal content, optimum pH, heat-sensitivity and stability were described.     

Cu-Zn-superoxide dismutase activity in Moniezia expansa: inhibition by pyrimidine derivatives

Copper-zinc, cyanide-sensitive superoxide dismutase (Cu-Zn-SOD) was detected in homogenates of Moniezia expansa. The enzyme was purified by a sequence of multiple differential centrifugations, ammonium sulphate precipitation, ion-exchange and G-75 Sephadex column chromatography. The final enzyme preparation had a specific activity of 623.00 +/- 9.97U per mg protein and, after isolation, a single-staining band on acrylamide-SDS gels was detected which coincided with enzyme activity. The inhibitory activities of several benzimidazoles and several novel pyrimidine derivatives were determined on purified extracts of the M. expansa Cu-Zn-SOD. The results indicated that the percentage inhibition of Cu-Zn-SOD by some pyrimidine derivatives (6-amino-1, 3-dimethyl-5-nitroso-uracil, 6-amino-5-methyl-5-nitroso-uracil and 5-amino-uracil) was markedly higher than inhibition with the benzimidazoles.     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

Effect of Zeitgeber Intensity Reduction on a Simulated Dual-Oscillator Human Circadian System: Classical and Dynamic Analysis

The two-oscillator model of human circadian rhythmicity was analyzed when a zeitgeber relative intensity of 1, 0.5, or 0.1 was introduced into the equations. Fourier analysis was compared with dynamic analysis such as attractor reconstruction or Liapunov exponent calculation. After a 50 or 90% reduction in zeitgeber intensity, the dynamics of the system became equivalent and differed significantly from those of a system with maximal zeitgeber intensity. When 10% aleatory noise was added to the data, the analysis was still applicable, and the results obtained were essentially the same as in the absence of noise. Dynamic analysis could thus provide a distinct classification for periodic data, based on the type of analysis.     

A semiempirical study of the prototropic tautomerism of hypoxanthine

The total and relative energies, bond order matrices and localized MOs for the eight possible tautomers of hypoxanthine (HYP) have been calculated, with full geometry optimization, using both AM1 and MNDO methods. The AM1 relative energies show that HYP(9,1), HYP(7,1) and HYP (9,10) are the predominant species at room temperature, the two former being in larger concentration that the latter. The calculated IR spectra for these species agree well with the reported spectrum in an isolated matrix, which has been interpreted in terms of the presence of these three tautomeric forms. The MNDO method does not predict the right order, and the more stable tautomer would be HYP(9,10). The calculated structure for the HYP(9,1) species shows that the molecule is essentially planar. The bond distances compare well with those of hypoxanthine hydrochloride and guanine and also correlate well with the calculated bond orders. The proton affinities for the three more stable tautomers have also been calculated. For HYP(9,1) the prefered site of protonation is N7, whereas for HYP(7,1) the protonation occurs rather at N9. These results agree well with¹⁵N and¹³C NMR studies in DMSO.