Catalytic and regulatory roles of species involved in metal–nucleotide equilibriums in human pyridoxal kinase

Pyridoxal 5′-phosphate is the active form of vitamin B₆ and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal–nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn²⁺ the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP^4? and HATP^3? did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP^2?, although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn²⁺; as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC₅₀ of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal–nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.     

ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.     

Potential Applications in Sewage Bioremediation of the Highly Efficient Pyridine-Transforming Paenochrobactrum sp.

Applied Biochemistry and Microbiology - A pyridine-transforming strain P2 was isolated from sewage collected from Guangzhou oil stain field(China).According to the system analysis, it was...     

Damming reduced the functional richness and caused the shift to a new functional state of the phytoplankton in a subtropical region

Hydrobiologia - We analyzed the effect of damming on the temporal patterns of the composition and richness of phytoplankton Reynolds functional groups (RFG) from a subtropical floodplain, together...     

Isolation of Candida in the blood cultures of patients admitted to the Emergency Room in a tertiary care hospital

BackgroundIn Spain, data of candidemia are limited to surveys conducted in specific areas or tertiary care centers. Also, in recent years, attention has shifted toward episodes of candidemia in non-ICU wards.AimsWe reviewed the cases of Candida isolates recovered from the blood of patients admitted to the Emergency Room (ER) in our tertiary care hospital.MethodsThe patients selected for this study had an isolation of Candida in the blood culture. All data were collected retrospectively from the clinical records of a 11-year period.ResultsCandida albicans and other species of the genus were present in 10 and 18 patients, respectively. The patients did not present different clinical features in comparison with other reports of hospitalized patients. All patients had several risk factors for candidemia. Only two patients had received previous antifungal therapy before admission. All the isolates of C. albicans, Candida glabrata and the only isolate of Candida tropicalis were susceptible to all the antifungal agents tested. Only one isolate of Candida parapsilosis was susceptible dose-dependent to fluconazole, and the only isolate of Candida metapsilosis was resistant to fluconazole.ConclusionsIt is essential to evaluate the risk factors, underlying conditions and clinical features in non-hospitalized patients in order to determine whether an empirical treatment for candidemia is appropriate.     

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

Plasma‐derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs‐derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean‐matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D‐DIGE)‐based study, and the other one for a label free LC–MS/MS‐based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty‐two and 23 differentially regulated features are detected from 2D‐DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D‐western‐blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma‐derived‐EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.     

Species diversity in Gymnogeophagus (Teleostei: Cichlidae) and comparative biogeography of cichlids in the Middle Paraná basin,an emerging hotspot of fish endemism

Hydrobiologia - We address the diversity of two species groups of the cichlid genus Gymnogeophagus in the Middle Paraná basin using molecular phylogeography and traditional morphological...     

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.