Biochemical characterization and gene structure analysis of the 24-kDa glutathione transferase sigma from Taenia solium.

Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease-control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long-term survival of T. solium; therefore, we structurally analyzed the 24-kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti-rTs24GST serum showed slight cross-reactivity with human sigma-class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G-site with GSH and of the H-site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis.

Eosinophils increase macrophage ability to control intracellular Leishmania amazonensis infection via PGD2 paracrine activity in vitro.

Clinical and experimental studies have described eosinophil infiltration in Leishmania amazonensis infection sites, positioning eosinophils strategically adjacent to the protozoan-infected macrophages in cutaneous leishmaniasis. Here, by co-culturing mouse eosinophils with L. amazonensis-infected macrophages, we studied the impact of eosinophils on macrophage ability to regulate intracellular L. amazonensis infection. Eosinophils prevented the increase in amastigote numbers within macrophages by a mechanism dependent on a paracrine activity mediated by eosinophil-derived prostaglandin (PG) D2 acting on DP2 receptors. Exogenous PGD2 mimicked eosinophil-mediated effect on managing L. amazonensis intracellular infection by macrophages and therefore may function as a complementary tool for therapeutic intervention in L. amazonensis-driven cutaneous leishmaniasis.

Leptin Elicits In Vivo Eosinophil Migration and Activation: Key Role of Mast Cell-Derived PGD2.

Eosinophils are key regulators of adipose tissue homeostasis, thus characterization of adipose tissue-related molecular factors capable of regulating eosinophil activity is of great interest. Leptin is known to directly activate eosinophils in vitro, but leptin ability of inducing in vivo eosinophilic inflammatory response remains elusive. Here, we show that leptin elicits eosinophil influx as well as its activation, characterized by increased lipid body biogenesis and LTC4 synthesis. Such leptin-triggered eosinophilic inflammatory response was shown to be dependent on activation of the mTOR signaling pathway, since it was (i) inhibited by rapamycin pre-treatment and (ii) reduced in PI3K-deficient mice. Local infiltration of activated eosinophils within leptin-driven inflammatory site was preceded by increased levels of classical mast cell-derived molecules, including TNFα, CCL5 (RANTES), and PGD2. Thus, mice were pre-treated with a mast cell degranulating agent compound 48/80 which was capable to impair leptin-induced PGD2 release, as well as eosinophil recruitment and activation. In agreement with an indirect mast cell-driven phenomenon, eosinophil accumulation induced by leptin was abolished in TNFR-1 deficient and also in HQL-79-pretreated mice, but not in mice pretreated with neutralizing antibodies against CCL5, indicating that both typical mast cell-driven signals TNFα and PGD2, but not CCL5, contribute to leptin-induced eosinophil influx. Distinctly, leptin-induced eosinophil lipid body (lipid droplet) assembly and LTC4 synthesis appears to depend on both PGD2 and CCL5, since both HQL-79 and anti-CCL5 treatments were able to inhibit these eosinophil activation markers. Altogether, our data show that leptin triggers eosinophilic inflammation in vivo via an indirect mechanism dependent on activation of resident mast cell secretory activity and mediation by TNFα, CCL5, and specially PGD2.

El rol de la proteína beta trace en distintas áreas de la Medicina / The role of beta trace protein in different areas of Medicine / O papel da proteína beta trace em diferentes áreas da Medicina

La proteína beta trace (PBT), también llamada Prostaglandina D2 Sintasa de tipo lipocalina, es una glicoproteína de peso molecular entre 23 y 29 kDa que convierte la prostaglandina H2 en prostaglandina D2. La misma está asociada a diferentes entidades clínicas. Por sus características moleculares puede ser un indicador útil de la alteración precoz en la filtración glomerular; por el aumento de su síntesis y su concomitante elevación en suero, un predictor de riesgo cardiovascular; y por su alta concentración en líquido cefalorraquídeo (LCR), un biomarcador de fístula de LCR. Puede medirse en distintos líquidos biológicos, como suero, orina y LCR. El objetivo de esta revisión fue actualizar los conocimientos de esta proteína para evaluar su utilidad en distintas áreas de la Medicina. La trascendencia de PBT en el campo de la bioquímica como posible biomarcador dependerá de la patología de base del paciente.

The beta trace protein (BTP), also called Prostaglandin D2 Synthase lipocalin type PGDS, is a glycoprotein between 23 and 29 kDa of low molecular weight that converts prostaglandin H2 in prostaglandin D2. BTP is a protein that has multiple clinical associations. Due to the characteristics of the molecule, it may indicate an early alteration in glomerular filtration; by the serum increase of its synthesis, it is a predictor of cardiovascular risk, and for its high concentration in cerebrospinal fluid (CSF), it is a marker of leakage. This protein can be measured in different biological fluids such as serum, urine, and CSF. The objective of this review is to update the knowledge about this protein as a biomarker. The significance of BTP in the field of biochemistry as a possible biomarker will depend on the patient's underlying pathology.

A proteína beta trace (PBT), também chamada de Prostaglandina D2, sintase de lipocalina é uma glicoproteína de peso molecular entre 23 e 29 kDa, que converte prostaglandina H2 em prostaglandina D2. PBT é uma proteína que está associada a várias entidades clínicas. Devido às características moleculares, pode ser uma indicação útil da alteração precoce na filtração glomerular, devido ao aumento de sua síntese e sua concomitante elevação em soro, um preditor de risco cardiovascular e, devido à sua alta concentração no líquido cefalorraquidiano (LCR), é um biomarcador da fístula LCR. É uma proteína que pode ser medida em diferentes fluidos biológicos, como soro, urina e LCR, e o objetivo desta revisão foi atualizar os conhecimentos desta proteína para avaliar sua utilidade em diversas áreas da medicina. A importância de PBT no campo da bioquímica como possível biomarcador dependerá da patologia subjacente do paciente.

Leptin Elicits LTC4 Synthesis by Eosinophils Mediated by Sequential Two-Step Autocrine Activation of CCR3 and PGD2 Receptors.

Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.

Participation of prostaglandin D2 in the mobilization of the nuclear-localized CTP:phosphocholine cytidylyltransferase alpha in renal epithelial cells.

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location.

Electroforesis bidimensional en orina: una alternativa para el laboratorio clínico / Two dimensional electrophoresis in urine: an alternative for the clinical laboratory / Eletroforese bidimensional em urina: uma alternativa para o laboratório clínico

En este trabajo se presenta un método confiable para el análisis de rutina de proteínas urinarias. El método es la electroforesis bidimensional que combina la electroforesis en acetato de celulosa con la electroforesis en gel de poliacrilamida con dodecil sulfato de sodio seguida de tinción argéntica. La electroforesis bidimensional abre el espectro de proteínas, siendo algunas de ellas identificadas por immunoblotting o espectrometría de masa MALDI. Dichas proteínas son de bajo peso molecular: Orosomucoide (40 kDa), apolipoproteína AI (28 kDa), zinc-alfa 2-glicoproteína (43 kDa), fragmento del perlecan C-terminal LG3 (23 kDa), prostaglandina D2 sintasa tipo-lipocalina (29 kDa) e inter-alfa inhibidor de tripsina cadena pesada H4 (35 kDa). Estas proteínas están incluidas en estudios de falla renal aguda, falla renal crónica, trasplante renal, enfermedad glomerular y enfermedad maligna del tracto urogenital. El método, no invasivo, se aproxima a la tecnología emergente de la proteómica que permite el análisis simultáneo de varias proteínas urinarias como una herramienta para el diagnóstico y monitoreo de una variedad de enfermedades humanas.(AU)

A method suitable for routine clinical analyses of urinary proteins is presented. This method is a two-dimensional electrophoresis procedure, combining cellulose acetate electrophoresis and sodium dodecyl sulfate-polyacrylamide gels electrophoresis followed by silver staining. The resulting two-dimensional electrophoresis opened the protein spectrum and some of the latter were identified, by immunoblotting or MALDI mass spectrometry. There are low molecular weight protein: Orosomucoid (40 kDa), apolipoprotein AI (28 kDa), Zinc-alpha2-glycoprotein (43 kDa), C-terminal perlecan fragment LG3 (23 kDa), lipocalin-type prostaglandin D2 synthase (29 kDa) and inter-alpha-trypsin inhibitor heavy chain H4 (35 kDa). They include studies of acute and chronic kidney injury, renal transplantation, glomerular disease and malignancy of the urogenital tract. This method approaching the emerging proteomic technologies allows simultaneous examination of the patterns of multiple urinary proteins as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases.(AU)

Neste trabalho é apresentado um método confiável para a análise de rotina de proteínas urinárias. O método é a eletroforese bidimensional que combina a eletroforese em acetato de celulose com a eletroforese em gel de poliacrilamida com dodecil sulfato de sódio seguida de tinþÒo argÛntea. A eletroforese bidimensional abre o espectro de proteínas, sendo algumas delas identificadas por immunoblotting ou espectrometria de massa MALDI. Tais proteínas sÒo de baixo peso molecular: Orosomucoide (40 kDa), apolipoproteína AI (28 kDa), zinco-alfa2-glicoproteína (43 kDa), fragmento do perlecan C-terminal LG3 (23 kDa), prostaglandina D2 sintase tipo-lipocalina (29 kDa) e inter-alfa inibidor de tripsina cadeia pesada H4 (35 kDa). Estas proteínas estÒo incluídas em estudos de falÛncia renal aguda, falÛncia renal cr¶nica, transplante renal, doenþa glomerular e doenþa maligna do trato urogenital. O método, nÒo invasivo, aproxima-se da tecnologia emergente da prote¶mica que permite a análise simultÔnea de várias proteínas urinárias como uma ferramenta para o diagnóstico e monitoraþÒo de uma variedade de doenþas humanas.(AU)