The heterologous expression of Escherichia coli MutT enzyme is involved in the protection against oxidative stress in Leishmania braziliensis

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.

Basophil Activation Test for Chronic Urticaria

No abstract available.

Estudio del sistema purinérgico a través de la caracterización de la enzima E-NPP3 y de la secreción de ATP en células uroepiteliales de la vejiga urinaria del ratón / Study of the purinergic system through the characterization of the E-NPP3 enzyme and the secretion of ATP in uroepithelial cells of the urinary bladder of the mouse

El presente trabajo involucra el estudio del sistema purinérgico mediante la purinérgico mediante la purificación y la caracterización bioquimica de la ecto-enzima E-NPP3 soluble, la cual modula la activación de los receptores purinérgicos mediante la hidrólisis de los nucleótidos extracelulares. La purificación de esta enzima, expresada en la línea celular CHO-K1, requirió el empleo de diferentes columnas cromatográficas; comprobándose la pureza, mediante la determinación de la actividad enzimática, la cuantificación de las proteínas y la detección de dicha enzima. Los resultados de la caracterización de la E-NPP3 indican que presentan un ph óptimo alcalino y que su actividad depende de la la concentración de los iones calcio y magnesio: mientras que el imidazol y el DDT ejercen acciones inhibidoras sobre su actividad. La purificación de la enzima E-NPP3 soluble representa un primer paso para futuros estudios que permitan su cristalización lo cual, constituirá una herramienta en la elaboración de agonistas y antagonistas selectivos o de anticuerpos monoclonales contra dicha enzima, útiles en el diagnóstico o el tratamiento de condiciones patológicas como el cáncer de colon y la colangiocarcinoma. Adicionalmente, teniendo en cuenta que se ha demostrado la disminución de la expresión de las ecto-nucleotidasas en el uroepitelio de pacientes que padecen cistitis intersticial; el presente trabajo comprende también, el desarrollo de un modelo de órgano aislado, la vejiga urinaria del ratón, para el estudio de la secreción de ATP desde las células uroepiteliales. Dicho modelo permitió demostrar, mediante estudios electrofisiológicos y el uso de diferentes fármacos, la contriución de los receptores purinérgicos sobre la actividad eléctrica del nervio pélvico cuando la vejiga es sometida a distensión mecánica gradual; sugiriendo la importancia de estos receptores en la transducción mecanosensorial de dicho órgano y permitiendo inferir la posible interrelación con los receptores de vaniloides en la detección de los estímulos sensoriales por parte del uroepitelio

Comparison of inosine triphate pyrophosphatase gene expression in chronic myelogenous leukemia patients and controls

One of the most significant damages to the genetic material is oxidative deamination of DNA and free nucleotides in the cell pool. Incorporation of deaminated purine nucleotides such as inosine triphosphate [ITP, dITP] into the DNA can increase the frequency of base substitution mutation. It has been suggested that presence and accumulation of these rough nucleotides can lead to genetic instability which is the perquisite of different types of diseases or cancers. Inosine triphosphaate pyrophosphates [ITPase/] encoded by ITPA gene, is responsible for protecting the cells by omitting deaminated purines from the free nucleotide pool. The objective of this study was to examine the possible dysfunction of ITPA gene activity as an important factor in genetic background predisposing to chromosomal disorders and malignancies such as CML. ITPA gene expression study performed on 23 CML patients and 21 controls using semi-quantitative RT-PCR technique and the expression level of GAPDH gene was used as an internal control. Unusual variants obtained from cDNA amplification of ITPA gene were clones. Our results showed a significant reduction of ITPA gene expression in CML patients in comparison to the controls. Two types of transcripts were produced in addition of expected transcript in some samples. One of them had a 123 nucleotide and the other had 77 nucleotide deletions in their open reading frame. Due to decreased expression of ITPA gene; it seems that the function of ITPase is not normal in CML patients. Therefore the altered expression of this gene can be considered as an additive factor for genetic instability in these patients

Estimation of postmortem intervals by using terminal deoxynucleotidyl transferase / 法医学杂志

OBJECTIVE@#To investigate the relationship between DNA degradeation and postmortem interval.@*METHODS@#dUTP was transferred to 3'terminal of DNA by using terminal deoxynucleotidyl transferase (TDT), then the reminders of dUTP after experimental reaction, as indicator of quantity of DNA degradation, were detected.@*RESULTS@#The reminders of dUTP were decreasing along with the postmortem interval.@*CONCLUSION@#Postmortem DNA degradation may be used in postmortem interval judgment.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80 similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme

Bioregulation of carbohydrate metabolism in relation to source-sink operation during grain-filling phase of growth in wheat.

Mobilization of free sugars from vegetative tissues to grain and their transformation to starch in relation to activities of some relevant enzymes during growth and development were investigated in wheat (Triticum aestivum L.). Vegetative tissues, viz. flag-leaf, flag-leaf sheath, nodes and internodes contained high concentration of free sugars from 70 DAS to 18 DPA and that was in the order of accumulation--flag-leaf sheath> flag-leaf and internodes > nodes. In these tissues, major portion of 14C appeared in endogenous sucrose, irrespective of the nature of (U-14C]-sugars supplied. In photosynthetic structures above flag-leaf node, namely peduncle, rachis and bracts, the free sugar make-up was maximum at anthesis (90 DAS). Activity of soluble acid invertase (EC 3.2.1.26) was high in these tissues during early stages of grain growth but reverse was true for soluble neutral invertase (EC 3.2.1.27) activity. In apical and basal portions of grain, free sugars were more or less similarly distributed in concentration. Linear and rapid accumulation of starch in endosperm paralleled with a decline in accumulation of this polymer in pericarp-aleurone. In the latter tissue, the activities of starch hydrolyzing enzymes, i.e alpha- and beta-amylases (3.2.1.1 and 3.2.1.2) were high during initial stages of grain growth. During active grain-filling, alkaline inorganic pyrophosphatase (EC 3.6.1.1) seemed to play a vital role during starch accumulation in endosperm, whereas the involvement of 3-PGA phosphatase (EC 3.1.3.38) was almost confined to pericarp-aleurone. Impairement of ear head photosynthesis by shading depressed starch synthesis (approximately 50%) indicating, thereby, the significant role of current photosynthates during grain-filling. The results suggested that grain growth in wheat was influenced by an efficient operation of source as well as regulatory factors, including enzymes, constituting intrinsic potential of grain sink.

Studies on the utilization of inorganic pyrophosphate by different metabolic systems in yeast mitochondria

A mitochondrial pyrophosphatase (PPase) from yeast cells (Saccharomyces cerevisiae) was studied and characterized. The hydrolytic activity towards inorganic pyrophosphate (PPi) was inhibited by different SH-reagents and increased in the presence of uncouplers, indicating a possible involvement of this enzyme in energy-linked processes. This view was also supported by the observation that these mitochondria were able to hydrolyze PPi, generating an electrical membrane potential (delta psi) of the same magnitude as that obtained with ATP. Both ATP and PPi inhibited the pyruvate dehydrogenase complex and it was demonstrated that PPi can be used as substrate by mitochondrial kinases leading to the same pattern of protein phosphorylation as when ATP is used