Inhibition of some hepatic glycosidases by the diseco nucleoside, 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analog

The in vivo and in vitro effects of 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analogue on alpha- and beta-glucosidases, beta-glucuronidase as well as alpha-amylase have been investigated. alpha-Glucosidase is the enzyme that is markedly affected in vivo and in vitro in a dose-dependent manner. The compounds showed a reversible inhibition of a competitive type for alpha-glucosidase. Moreover, they exert a relatively potent inhibition on alpha-glucosidase with a Ki magnitude of 3.6 x 10(-4), 9.5 x 10(-5) M.     

Global Metabolomic Profiling of Mice Brains following Experimental Infection with the Cyst-Forming Toxoplasma gondii

The interplay between the Apicomplexan parasite Toxoplasma gondii and its host has been largely studied. However, molecular changes at the metabolic level in the host central nervous system and pathogenesis-associated metabolites during brain infection are largely unexplored. We used a global metabolomics strategy to identify differentially regulated metabolites and affected metabolic pathways in BALB/c mice during infection with T. gondii Pru strain at 7, 14 and 21 days post-infection (DPI). The non-targeted Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics analysis detected approximately 2,755 retention time-exact mass pairs, of which more than 60 had significantly differential profiles at different stages of infection. These include amino acids, organic acids, carbohydrates, fatty acids, and vitamins. The biological significance of these metabolites is discussed. Principal Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis showed the metabolites’ profile to change over time with the most significant changes occurring at 14 DPI. Correlated metabolic pathway imbalances were observed in carbohydrate metabolism, lipid metabolism, energetic metabolism and fatty acid oxidation. Eight metabolites correlated with the physical recovery from infection-caused illness were identified. These findings indicate that global metabolomics adopted in this study is a sensitive approach for detecting metabolic alterations in T. gondii-infected mice and generated a comparative metabolic profile of brain tissue distinguishing infected from non-infected host.     

Antibiotic susceptibility and genotype patterns of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from urinary tract infected patients

Thirty nine isolates of Escherichia coli, twenty two isolates of Klebsiella pneumoniae and sixteen isolates of Pseudomonas aeruginosa isolated from urinary tract infected patients were analyzed by antimicrobial susceptibility typing and random amplified polymorphic DNA (RAPD)-PCR. Antibiotic susceptibility testing was carried out by microdilution and E Test methods. From the antibiotic susceptibility, ten patterns were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively). Furthermore, genotyping showed seventeen RAPD patterns (seven for E. coli, five for K. pneumoniae and five for P. aeruginosa respectively). In this study, differentiation of strains of E. coli, K. pneumoniae and P. aeruginosa from nosocomial infection was possible with the use of RAPD.     

Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress

The present study aimed to evaluate the association between the environmental tobacco smoke (ETS) and DNA damage in relation to oxidative stress (OS) in children. Sixty-four children of age 1-8 years, selected from the outpatient clinic of Mansoura University Children Hospital were divided into two groups (23 children/group) based on high (>20 cigarettes/day) or low (<20 cigarettes/day) exposure to ETS at home. Twenty symptom-free children with normal cotinine level and with no exposure to ETS were recruited as controls. The comet assay was used to quantify the level of DNA damage in lymphocytes isolated from all children. Spectrophotometric methods were used to assess the serum level of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-Px) in erythrocytes. Also, serum level of tocopherol fractions (alpha, gamma, delta) was assessed by high performance liquid chromatography (HPLC). Children exposed to ETS exhibited retarded growth, more chest problems, and gastroenteritis than the control. A significant increase in mean comet tail length indicating DNA damage was observed in ETS-exposed children (P<0.001) compared to controls. ETS-exposed children had significantly (P<0.001) higher MDA level paralleled with significant (P<0.001) decrease in the level of GSH-Px and tocopherol fractions compared with controls. The GSH-Px activity and tocopherol levels were inversely correlated with the increase of ETS exposure. These results show that inhalation of ETS is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in the children's blood. This status of oxidant-antioxidant imbalance (OS) may be one of the mechanisms leading to DNA damage detected in lymphocytes of ETS-exposed children. In conclusion, the present study gives an indication of an association between DNA damage and ETS exposure in children.     

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.     

SDS-PAGE heat-shock protein profiles of environmental Aeromonas strains

Aeromonas microorganisms normally grow at temperatures between 5 degrees C and 45 degrees C and therefore should have high thermotolerance. Thus it was of interest to find out whether A. hydrophila, A. caviae and A. veronii biovar sobria serovars respond to abrupt temperature changes with a heat shock-like response. To this end the present study was undertaken to determine whether Aeromonas species exhibits a heat shock response to different temperatures and time factors. The response of Aeromonas serovars to 24 h and 48 h of thermal stress at 25 degrees C, 42 degrees C and 50 degrees C involved the synthesis of 12-18 heat shock proteins (HSPs) bands with molecular weights ranging between 83.5-103.9 kDa in the high HSP molecular mass and 14.5-12.0 as low molecular mass HSP. Electrophoretic analysis of the HSPs showed that the serovars do not cluster very tightly and also that they are distinct from each other.     

Phylogenetic congruence of <Emphasis Type="Italic">Sarcocystis neurona</Emphasis> Dubey et al., 1991 (Apicomplexa: Sarcocystidae) in the United States based on sequence analysis and restriction fragment length polymorphism (RFLP)

The objectives of the present study were to assess the genetic diversity, phylogeny and phylogeographical relationships of available Sarcocystis neurona isolates from different localities in the United States. All 13 Sarcocystis isolates from different hosts were subjected to polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses using two published DNA markers (25/396 and 33/54). The 334 bp sequence of the 25/396 marker of these isolates and Besnoitia darlingi, B. bennetti, Toxoplasma gondii and Neospora caninum were sequenced and compared. Phylogenetic analysis was performed using neighbour-joining (NJ), maximum parsimony (MP) and minimum evolution (ME) methods based on the sequences of the 25/396 marker of the 13 Sarcocystis isolates obtained in this study and sequences of 10 related isolates from GenBank. Phylogenetic trees revealed a close relatedness among S. neurona isolates in the US (nucleotide sequence diversity <5.0%). US isolates formed a monophyletic group and appeared more closely related to each other than to the South American isolates, which formed a separate lineage. NJ and ME trees with Kimura 2-parameter model separated S. neurona into two separate groups: a northern US group and a Southern US group. These findings suggest a correlation between grouping of the isolates and geographical segregation and were consistent with a genetic bottleneck hypothesis during opossum colonisation of North America. These data do not support either the view of S. neurona as a single super-species or its division into multiple subspecies.