HGPRT and PNP: Recombinant Enzymes from Schistosoma mansoni and Their Role in Immunotherapy during Experimental Murine Schistosomiasis.

Schistosomiasis is a parasitic infection caused by trematode worms (also called blood flukes) of the genus Schistosoma sp., which affects over 230 million people worldwide, causing 200,000 deaths annually. There is no vaccine or new drugs available, which represents a worrying aspect, since there is loss of sensitivity of the parasite to the medication recommended by the World Health Organization, Praziquantel. The present study evaluated the effects of the recombinant enzymes of S. mansoni Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT), Purine Nucleoside Phosphorylase (PNP) and the MIX of both enzymes in the immunotherapy of schistosomiasis in murine model. These enzymes are part of the purine salvage pathway, the only metabolic pathway present in the parasite for this purpose, being essential for the synthesis of DNA and RNA. Female mice of Swiss and BALB/c strains were infected with cercariae and treated, intraperitoneally, with three doses of 100 µg of enzymes. After the immunotherapy, the eggs and adult worms were counted in the feces; the number of eosinophils from the fluid in the peritoneal cavity and peripheral blood was observed; and the quantification of the cytokine IL-4 and the production of antibodies IgE was analyzed. The evaluation of the number of granulomas and collagen deposition via histological slides of the liver was performed. The results demonstrate that immunotherapy with the enzyme HGPRT seems to stimulate the production of IL-4 and promoted a significant reduction of granulomas in the liver in treated animals. The treatment with the enzyme PNP and the MIX was able to reduce the number of worms in the liver and in the mesenteric vessels of the intestine, to reduce the number of eggs in the feces and to negatively modulate the number of eosinophils. Therefore, immunotherapy with the recombinant enzymes of S. mansoni HGPRT and PNP might contribute to the control and reduction of the pathophysiological aspects of schistosomiasis, helping to decrease the morbidity associated with the infection in murine model.

In vitro and in vivo characterization of the multiple isoforms of Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferases.

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.

Lesch-Nyhan disease in two families from Chiloé Island with mutations in the HPRT1 gene.

Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report two independent point mutations leading to splicing errors: IVS 2 +1G>A, c.134 +1G>A, and IVS 3 +1G>A, c.318 +1G>A in the hypoxanthine-phosphoribosyltransferase1 (HPRT1) gene which result in exclusion of exon 2 and exon 3 respectively, in the HGprt enzyme protein from different members of two Chiloé Island families. Molecular analysis has revealed the heterogeneity of genetic mutation of the HPRT1 gene responsible for the HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.

Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

Functional food investigations have demonstrated the presence of substances that could be beneficial to human health when consumed. However, the toxic effects of some substances contained in foods have been determined. Reported medicinal and nutritive properties have led to the extensive commercialization of the basidiomycete fungi Agaricus blazei Murrill (sensu Heinemann), also known as Agaricus brasiliensis Wasser et al., Agaricus subrufescens Peck or the Brazilian medical mushroom (BMM). Different methanolic extract fractions (ME) of this mushroom were submitted to the cytokinesis-block micronucleus (CBMN) clastogenic assay and the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT) assay for gene mutation, both using Chinese hamster ovary cells clone K1 (CHO-K1). The results suggest that all the fractions tested possess cytotoxic and mutagenic potential but no clastogenic effects. Further information is needed on the biochemical components of the A. blazei methanol fractions to identify any substances with cytotoxic and/or mutagenicity potential. These findings indicate that A. blazei methanolic extract should not be used due to their genotoxicity and care should be taken in the use of A. blazei by the general population until further biochemical characterization of this fungi is completed.

Determinación de las constantes cinéticas de la enzima hipoxantina - guanina fosforribosiltransferasa (HGPRT) en controles normales y en una familia afectada por el Sindrome de Lesch-Nyhan / Determination of the Kinetic constants of hypoxanthine guanine phosphoribosyl transférase enzyme (HGPRT) in normal controls and in a family affected by Lesch-Nyhan’s syndrome

El sindrome de Lesch-Nyhan (SLN) es producido por una deficiencia total de la enzima Hipoxantina-Guanina fosforribosiltransferasa (HGPRT). Las madres de niños afectados por el SLN son heterocigotas obligadas ya que la enfermedad se hereda de forma recesiva ligada al cromosoma X. Una de las células utilizadas para determinar la enfermedad es el eritrocito; sin embargo, la determinación de la condición portadora de las madres con niños afectados no se puede hacer en estas células ya que presentan una actividad que cae dentro del rango considerado normal.Esto sucede porque el eritrocito deficiente en la enzima HGPRT es destruido antes de que alcance la circulación sanguínea. Aplicando los principios cinéticos de Lineweaver-Burk y mediante espectrofotometría se determino la cinética de la (HGPRT) extraída de los eritrocitos de una familia que sufre el Sindrome de Lesch-Nyhan y se compararon con la cinética de esta enzima utilizando eritrocitos de 10 individuos sanos y normales procesados de igual forma. Los dos sustratos estudiados fueron la Guanina y el Fosforribosilpirofosfato (PRPP). La Guanina en individuos normales presentó un rango de Vmax entre 1.7 a 2.8 μmol de GMP/ min/ g Hb. mientras que el rango presentado para la Km fue de 10 a 25 μM. El PRPP en los controles normales presentó un rango para la Vmax de 2 a 2.7 μmol de GMP/min/g Hb y el rango para la Km fue de 301 a 590 μM. estos valores corresponden a lo reportado por otros autores. En la familia estudiada la cual tiene dos niños que padecen el síndrome de Lesch- Nyhan, tanto el padre como la hija presentaron cinéticas que caen dentro del rango considerado normal, mientras que la madre presentó una alteración en la Km para el PRPP ( 1176 μ M ). Cuando se comparó la regresión linear de las pendientes presentadas por los controles con la presentada por la paciente portadora de la enfermedad se encontró que estadísticamente son muy diferentes, diferencia que es ocasionada por el valor elevado de la Km de esta paciente por el sustrato PRPP.

The syndrome of Lesch-Nyhan (SLN) is produced by total deficiency of the enzyme Hipoxanthine-Guanine Phosphoribosyltransferase (HGPRT). The mothers of children affected by the SLN are obliged heterocigotes since the illness is inherited from a recessive way bound to the chromosome X. One of the utilized cells to diagnostic the illness is the erythrocyte; however, the determination of the condition carrier of the mothers with affected children cannot make in these cells since they present an activity that falls inside the normal considered range. This happens because the faulty erythrocyte in the enzyme HGPRT is destroyed before it reaches the blood stream. Applying the kinetic principles of Lineweaver-Burk and by means of espectrophotometry I to carry out the kinetic of the HGPRT enzyme extracted of the erythrocytes of a family that it suffers the SLN and they were compared with the kinetics of this enzyme in erythrocytes of ten individuals health and normal. The two studied substrates were the Guanine and the phosphorybosylpirophosphate(PRPP). The Guanine in normal individuals presented a range of Vmax among 1.7 at 2.8 μmol GMP/min/gHb, while the range presented for the Km went from 10 to 25 μ M. The PRPP in the normal controls presented a range for the Vmax from 2 at 2.7μmol GMP/min/gHb and the range for the Km went from 301 to 590 μM. These ranges are similar to those reported by other authors. In the estudied family which has two children that suffer the SLN , as much the father as the daughter presented kinetic that fall inside the normal considered range, while the mother presented increased the km for the PRPP ( the value of Km for the PRPP of the mother of the children affected by the syndrome was of 1176 μ M. When we compared the pendent the normal controls with the obliged heterocigotes pendent for the PRPP substrate was statisticaly different owing to high Km value.