Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme

THERAPEUTIC APPROACH TO A CHILD WITH ACUTE RESPIRATORY DISTRESS SYNDROME: A REPORT OF TWO CASES

The course of a respiratory disorder in a child may end up in respiratory failure. There are also acute non-respiratory diseases which have a great influence on the respiratory functions and often lead to the acute lung injury and sometimes to the acute respiratory distress syndrome (ARDS). A feature of respiratory function deterioration is changed in the surfactant system. We often see inhibition of its synthesis or damage to its structure. Therapy of children suffering from ARDS should be complex and rapid. Despite many recently published studies explaining the principle of this disorder, the mortality of ARDS is still very high (30-50%). There are several studies documenting successful administration of exogenous surfactant as part of a complex combined therapy of patients with ARDS, which leads to decreased mortality, improved oxygenation, and decreased need for aggressive artificial pulmonary ventilation. The authors of this article present their own experience with administration of exogenous surfactant in therapy of children with ARDS

Effects of Methionine Gamma Lyase-Deaminase on Human Colorectal Carcinoma Cells

Objective. The objective of this study is to demonstrate the effects of methionine gamma lyase-deaminase (Mgld) on colorectal carcinoma cell survival using MTT assay. Background. Mgld is in enzyme found in bacteria and protozoa including, Porphyromonas gingivalis. Mgld has the ability to catabolize methionine, an enzyme function that is absent in mammals. This enzyme function is of interest in methionine/S-adenosyl methionine (SAM) dependent cancer cells given the dysregulation of DNA methylation patterns that occur compared to normal mammalian cells. Methods. In this study, Porphyromonas gingivalis Mgld cloned into a cytoplasmic and nuclear plasmid vector was transfected into the human colorectal carcinoma T84 cell line using Lipofectamine 3000. The effects of cytoplasmic Mgld and nuclear Mgld were assessed in comparison to control non-transfected cells. Cell survival was assessed with an MTT assay at 570 nm as an indicator of metabolic functions of the live cells/mitochondria. Results. Results indicate that nuclear Mgld transfection causes most significant inhibition of metabolic activity in T84 colorectal carcinoma cells with a 19% decrease in absorbance compared to the control. Conclusion. Due to its effects on cellular survival, further studies should be conducted to evaluate and investigate the metabolic implications of nuclear Mgld on T84 colorectal carcinoma cells

One Session Bilateral Carotid Stenting – Experience from Slovakian Center

Objectives: Bilateral carotid artery stenting (BCAS) in one session is relatively rare procedure. However, clear recommendations still do not exist. The aim of our study was to analyze efficacy and safety of the BCAS performed by single operator in our center