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.     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.     

A theoretical and experimental study of ionic shifts induced by K depletion and replacement.

The mutual synchronization or entrainment of inter-connected self-oscillatory systems is relevant in a number of biological models. It forms the basis of a currently-accepted model for the electrical “slow-wave” activity in the mammalian gastro-intestinal tract, and is of interest in oscillator population studies for circadian rhythm research. In this paper, conditions of almost-entrainment when periodic fluctuations in amplitude and/or frequency occur are analysed using the method of harmonic balance. The effects of oscillator non-linearity and different types of coupling component are demonstrated, and results are compared with spectral analysis of simulated oscillators.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Rapid detection of HSV from cytologic specimens collected into ThinPrep fixative

OBJECTIVE: Herpes simplex virus (HSV) infection is associated with substantial morbidity and mortality in neonates. A diagnosis of HSV on cervical cytologic studies could lead to a cesarean section, with an increase in the risk of maternal morbidity. The identification of viral lesions in sexually active women has medical and social implications. There have been reports of false positive diagnoses of HSV in patients with altered endocervical cells and with cervical intraepithelial neoplasia 3. We evaluated a polymerase chain reaction (PCR)-based assay to detect HSV-1 and HSV-2 in routinely collected cervical cytology specimens in ThinPrep fixative (Cytyc Corp., Marlborough, Massachussets, U.S.A.). STUDY DESIGN: DNA was extracted from five cases that demonstrated cytologic changes suggestive of an HSV infection. PCR amplification with subsequent gel electrophoresis was performed to detect the presence of HSV. RESULTS: HSV DNA was detected in three of five cases that were cytologically diagnosed as suspicious or strongly suspicious for HSV infection. CONCLUSION: The combination of the ThinPrep liquid-based method for cervical cytology with PCR allows prompt confirmation of the diagnosis of HSV without sacrificing the diagnostic morphology on the slide.     

Plant defence as a complex and changing phenotype throughout ontogeny

Background and Aims Ontogenetic changes in anti-herbivore defences are common and result from variation in resource availability and herbivore damage throughout plant development. However, little is known about the simultaneous changes of multiple defences across the entire development of plants, and how such changes affect plant damage in the field. The aim of this study was to assess if changes in the major types of plant resistance and tolerance can explain natural herbivore damage throughout plant ontogeny.Methods An assessment was made of how six defensive traits, including physical, chemical and biotic resistance, simultaneously change across the major transitions of plant development, from seedlings to reproductive stages of Turnera velutina growing in the greenhouse. In addition, an experiment was performed to assess how plant tolerance to artificial damage to leaves changed throughout ontogeny. Finally, leaf damage by herbivores was evaluated in a natural population.Key Results The observed ontogenetic trajectories of all defences were significantly different, sometimes showing opposite directions of change. Whereas trichome density, leaf toughness, extrafloral nectary abundance and nectar production increased, hydrogen cyanide and compensatory responses decreased throughout plant development, from seedlings to reproductive plants. Only water content was higher at the intermediate juvenile ontogenetic stages. Surveys in a natural population over 3 years showed that herbivores consumed more tissue from juvenile plants than from younger seedlings or older reproductive plants. This is consistent with the fact that juvenile plants were the least defended stage.Conclusions The results suggest that defensive trajectories are a mixed result of predictions by the Optimal Defence Theory and the Growth–Differentiation Balance Hypothesis. The study emphasizes the importance of incorporating multiple defences and plant ontogeny into further studies for a more comprehensive understanding of plant defence evolution.     

MMP-13 selective α-sulfone hydroxamates: a survey of P1' heterocyclic amide isosteres

Seeking compounds preferentially potent and selective for MMP-13, we reported in the preceding Letter on a series of hydroxamic acids with a flexible benzamide tail groups.^1a Here, we replace the amide moiety with non-hydrolyzable heterocycles in an effort to improve half-life. We identify a hydroxamate tetrazole 4e that spares MMP-1 and -14, shows >400-fold selectivity versus MMP-8 and >600-fold selectivity versus MMP-2, and has a 4.8 h half-life in rats. X-ray data (1.9 Å) for tetrazole 4c is presented.     

Effects of inhaled corticosteroids on sputum cell counts in stable chronic obstructive pulmonary disease: a systematic review and a meta-analysis

Background

Whether inhaled corticosteroids suppress airway inflammation in chronic obstructive pulmonary disease (COPD) remains controversial. We sought to determine the effects of inhaled corticosteroids on sputum indices of inflammation in stable COPD.

Methods

We searched MEDLINE, EMBASE, CINAHL, and the Cochrane Databases for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled corticosteroids in stable COPD. For each chosen study, we calculated the mean differences in the concentrations of sputum cells before and after treatment in both intervention and control groups. These values were then converted into standardized mean differences to accommodate the differences in patient selection, clinical treatment, and biochemical procedures that were employed across original studies. If significant heterogeneity was present (p < 0.10), then a random effects model was used to pool the original data. In the absence of significant heterogeneity, a fixed effects model was used.

Results

We identified six original studies that met the inclusion criteria (N = 162 participants). In studies with higher cumulative dose (≥ 60 mg) or longer duration of therapy (≥ 6 weeks), inhaled corticosteroids were uniformly effective in reducing the total cell, neutrophil, and lymphocyte counts. In contrast, studies with lower cumulative dose (< 60 mg) or shorter duration of therapy (< 6 weeks) did not demonstrate a favorable effect of inhaled corticosteroids on these sputum indices.

Conclusions

Our study suggests that prolonged therapy with inhaled corticosteroids is effective in reducing airway inflammation in stable COPD.