Nephroprotective effect of pioglitazone in a Wistar rat model of adenine­induced chronic kidney disease.

Chronic kidney disease (CKD) is a progressive disease with a high mortality rate and a worldwide prevalence of 13.4%, triggered by various diseases with high incidence. The aim of the present study was to investigate the anti-inflammatory and antifibrotic effect of pioglitazone on kidney in an adenine-induced Wistar rats and the mechanisms possibly involved. CKD was induced in 40 rats. Rats were divided into two groups, which were split into the following sub-groups: i) Therapeutic (pioglitazone administered after renal damage) divided into intact (healthy), adenine (CKD) and adenine/pioglitazone (treatment) and ii) prophylactic (adenine and pioglitazone administered at the same time) split into intact (healthy), adenine (CKD), endogenous reversion (recovery without treatment), adenine/pioglitazone (treatment) and pioglitazone sub-groups. Reverse transcription-quantitative PCR (collagen I, α-SMA and TGF-ß), and hematoxylin-eosin, Masson's trichrome and Sirius red staining were performed to measure histological markers of kidney damage, also the serum markers (urea, creatinine and uric acid) were performed, for analyze the effects of pioglitazone. In the adenine/pioglitazone rats of the therapeutic group, renal function parameters such as eGFR increased and serum creatinine decreased from those of untreated rats (CKD), however the renal index, serum urea, abnormalities in renal morphology, inflammatory cells and relative gene expression of collagen I, α-SMA and TGF-ß did not change relative to the CKD rats. In adenine/pioglitazone rats, extracellular matrix collagen accumulation was significantly lower than the CKD rats. On the other hand, in adenine/pioglitazone rats of the prophylactic group, the renal index, creatinine, urea, uric acid serum and relative gene expression of collagen I, α-SMA, and TGF-ß were significantly lower, as well as the presence of 2,8-dihydroxyadenine crystals, and extracellular matrix collagen compared with CKD rats. In addition, the eGFR in the treatment group was similar to healthy rats, renal morphology was restored, and inflammatory cells were significantly lower. In conclusion, pioglitazone has a nephroprotective effect when administered in the early stages of kidney damage, reducing inflammatory and fibrotic processes and improving glomerular filtration rate. Furthermore, in the late phase of treatment, a tendency to decrease creatinine and increase eGFR was observed.

The Pigment World: Life's Origins as Photon-Dissipating Pigments.

Many of the fundamental molecules of life share extraordinary pigment-like optical properties in the long-wavelength UV-C spectral region. These include strong photon absorption and rapid (sub-pico-second) dissipation of the induced electronic excitation energy into heat through peaked conical intersections. These properties have been attributed to a "natural selection" of molecules resistant to the dangerous UV-C light incident on Earth's surface during the Archean. In contrast, the "thermodynamic dissipation theory for the origin of life" argues that, far from being detrimental, UV-C light was, in fact, the thermodynamic potential driving the dissipative structuring of life at its origin. The optical properties were thus the thermodynamic "design goals" of microscopic dissipative structuring of organic UV-C pigments, today known as the "fundamental molecules of life", from common precursors under this light. This "UV-C Pigment World" evolved towards greater solar photon dissipation through more complex dissipative structuring pathways, eventually producing visible pigments to dissipate less energetic, but higher intensity, visible photons up to wavelengths of the "red edge". The propagation and dispersal of organic pigments, catalyzed by animals, and their coupling with abiotic dissipative processes, such as the water cycle, culminated in the apex photon dissipative structure, today's biosphere.

The essential role of adenine nucleotide translocase 4 on male reproductive function in mice

Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.

Infrared Spectral Signatures of Nucleobases in Interstellar Ices I: Purines.

The purine nucleobases adenine and guanine are complex organic molecules that are essential for life. Despite their ubiquitous presence on Earth, purines have yet to be detected in observations of astronomical environments. This work therefore proposes to study the infrared spectra of purines linked to terrestrial biochemical processes under conditions analogous to those found in the interstellar medium. The infrared spectra of adenine and guanine, both in neat form and embedded within an ice made of H2O:NH3:CH4:CO:CH3OH (10:1:1:1:1), were analysed with the aim of determining which bands attributable to adenine and/or guanine can be observed in the infrared spectrum of an astrophysical ice analogue rich in other volatile species known to be abundant in dense molecular clouds. The spectrum of adenine and guanine mixed together was also analysed. This study has identified three purine nucleobase infrared absorption bands that do not overlap with bands attributable to the volatiles that are ubiquitous in the dense interstellar medium. Therefore, these three bands, which are located at 1255, 940, and 878 cm-1, are proposed as an infrared spectral signature for adenine, guanine, or a mixture of these molecules in astrophysical ices. All three bands have integrated molar absorptivity values (ψ) greater than 4 km mol-1, meaning that they should be readily observable in astronomical targets. Therefore, if these three bands were to be observed together in the same target, then it is possible to propose the presence of a purine molecule (i.e., adenine or guanine) there.

Enhanced production of cordycepin under solid-state fermentation of Cordyceps militaris by using combinations of grains/substrates.

This manuscript deals with cordycepin, an interesting secondary compound produced from entomopathogenic fungus, Cordyceps. It has attracted commercial interest due to its immense pharmacological importance beneficial to human health. In this study, the contents of cordycepin and its derivatives, like adenine and adenosine, were evaluated through solid-state fermentation using combinations of various grains as substrate. Treatment with grain combination numbers 2, 7, 8, and 9 exhibited higher cordycepin content (1.621, 1.929, 1.895, and 1.996 mg/g cordycepin, respectively) than control (rice). The grain combination number 7 exhibited significantly higher adenine content (700 mg/g) than the control and all other combinations. Treatments with grain combination numbers 2, 5, and 7 exhibited higher adenosine content (2.719, 2.938, and 3.392 mg/g, respectively); however, no significant increase in adenosine content was noted in any treatments. The biomass including fresh mycelium and fruit body was found higher in grain combination numbers 7 and 9, leading to enhanced cordycepin content. Overall, the increase in the fresh biomass significantly enhanced cordycepin accumulation. The level of cordycepin was recorded as higher than that of its derivatives, adenosine and adenine. The grain combination of rice, wheat, jowar, bajra, and sugarcane bagasse added to basal medium exhibited the highest cordycepin content and was found suitable for solid-state fermentation of Cordyceps militaris. To our understanding, the present study is the first to use combinations of cereals for the production of cordycepin from C. militaris.

DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.

Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants' adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently "touched" by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.

The Non-Equilibrium Thermodynamics of Natural Selection: From Molecules to the Biosphere.

Evolutionary theory suggests that the origin, persistence, and evolution of biology is driven by the "natural selection" of characteristics improving the differential reproductive success of the organism in the given environment. The theory, however, lacks physical foundation, and, therefore, at best, can only be considered a heuristic narrative, of some utility for assimilating the biological and paleontological data at the level of the organism. On deeper analysis, it becomes apparent that this narrative is plagued with problems and paradoxes. Alternatively, non-equilibrium thermodynamic theory, derived from physical law, provides a physical foundation for describing material interaction with its environment at all scales. Here we describe a "natural thermodynamic selection" of characteristics of structures (or processes), based stochastically on increases in the global rate of dissipation of the prevailing solar spectrum. Different mechanisms of thermodynamic selection are delineated for the different biotic-abiotic levels, from the molecular level at the origin of life, up to the level of the present biosphere with non-linear coupling of biotic and abiotic processes. At the levels of the organism and the biosphere, the non-equilibrium thermodynamic description of evolution resembles, respectively, the Darwinian and Gaia descriptions, although the underlying mechanisms and the objective function of selection are fundamentally very different.

Adenine phosphoribosyl transferase deficiency leads to renal allograft dysfunction in kidney transplant recipients: a systematic review / A deficiência de adenina fosforibosiltransferase leva à disfunção do aloenxerto renal em receptores de transplante renal: uma revisão sistemática

Abstract Background: Adenine phosphoribosyl transferase (APRT) deficiency has great implications on graft survival in kidney transplant patients. This systematic review investigated the diagnostic pattern, treatment approach, and kidney transplant outcomes among kidney transplant patients with adenine phosphoribosyl transferase deficiency. Material and methods: Articles reporting the APRT enzyme deficiency and kidney allograft dysfunction were retrieved from PubMed/Medline, ScienceDirect, Cochrane library and Google scholar databases. Descriptive analysis was used to draw inferences. Results: The results from 20 selected studies covering 30 patients receiving 39 grafts had an average age of 46.37 years are presented. Graft survival time of more than 6 months was reported in 23 (76.7%) patients, while other 7 (23.3%) patients had graft survival time of less than 6 months. Only 4 (13.3%) patients had APRT deficiency before transplantation. After follow-up, one-third of the patients 10 (33.3%) had stable graft function, 1 patient had allograft loss, 8 (26.6%) patients had delayed graft function while the remaining 11 (36.6%) patients had chronic kidney graft dysfunction. Conclusions: APRT deficiency is an under-recognized, treatable condition that causes reversible crystalline nephropathy, leading to loss of allograft or allograft dysfunction. The study results showed that inclusion of genetic determination of APRT deficiency in the differential diagnosis of crystalline nephropathy, even in the absence of a history of nephrolithiasis, can improve renal outcomes and may improve allograft survival.

Resumo Antecedentes: A deficiência de adenina fosforibosiltransferase (APRT) tem grandes implicações na sobrevida do enxerto em pacientes transplantados renais. Esta revisão sistemática investigou o padrão diagnóstico, a abordagem de tratamento e os desfechos do transplante renal entre pacientes transplantados renais com deficiência de adenina fosforibosiltransferase. Material e métodos: Os artigos que relatam sobre a enzima APRT e a disfunção do aloenxerto renal foram recuperados do PubMed/Medline, ScienceDirect, Biblioteca Cochrane e bancos de dados do Google Acadêmico. Utilizou-se a análise descritiva para extrair inferências. Resultados: Foram incluídos participantes que receberam 39 enxertos, a maioria dos quais provenientes de doadores vivos seguidos por doadores falecidos e doadores cadáveres. Foi relatado tempo de sobrevida do enxerto superior a 6 meses em 23 (76,7%) pacientes, enquanto outros 7 (23,3%) pacientes tiveram tempo de sobrevida do enxerto inferior a 6 meses. Apenas 4 (13,3%) pacientes apresentaram deficiência de APRT antes do transplante. Após acompanhamento, um terço dos pacientes, 10 (33,3%) apresentaram função do enxerto estável, 1 paciente teve perda do aloenxerto, 8 (26,6%) pacientes apresentaram função retardada do enxerto, enquanto os 11 (36,6%) pacientes restantes tiveram disfunção crônica do enxerto renal. Conclusões: A deficiência de APRT é uma causa subestimada e reversível de nefropatia cristalina que leva à disfunção do aloenxerto renal ou à perda total do aloenxerto. Os resultados deste estudo pedem a inclusão desta condição no diagnóstico diferencial de nefropatia cristalina, mesmo na ausência de um histórico de nefrolitíase.

Orchiectomy but not adjuvant-induced arthritis induces structural modifications in rat aortas.

PURPOSE: This study aimed to verify whether Adjuvant-Induced Arthritis (AIA) and/or Orchiectomy (ORX) modify the expression of the Nox1, Nox2 and Nox4 isoforms, the endothelial function or the structure of rat aortas. METHODS: Sixty-three Wistar rats were distributed into four groups: 1) Control; 2) ORX; 3) AIA; 4) Orchiectomy plus to Arthritis-induction (ORX/AIA). Thus, 21 days after the onset of AIA (by intradermal injection of Mycobacterium tuberculosis), the presence of Nox1, Nox2 and Nox4, the acetylcholine (ACh)-induced relaxation and the media layer thickness were assessed in the aorta taken from these animals. RESULTS: The Nox1, Nox2 and Nox4 were immunostained in intima, media and adventitia layers of aortas taken from all studied groups and AIA apparently increased this immunostaining. These modifications of Nox1, Nox2 or Nox4 expression, however, were not confirmed by Western blotting. In addition, neither AIA nor ORX changed the endothelial function, but ORX increased the media layer thickness in the studied aortas. CONCLUSION: The present study showed weak clues of increased expression of Nox1, Nox2 and Nox4 as a result of AIA, as well as of Nox1 reduction caused by ORX. In addition, the endothelial function was not modified in the aortas of these animals by both AIA and/or ORX. On the other hand, ORX increased significantly the aorta media layer thickness in the studied animals, which was apparently mitigated by AIA.

Dissipative Photochemical Abiogenesis of the Purines.

We have proposed that the abiogenesis of life around the beginning of the Archean may have been an example of "spontaneous" microscopic dissipative structuring of UV-C pigments under the prevailing surface ultraviolet solar spectrum. The thermodynamic function of these Archean pigments (the "fundamental molecules of life"), as for the visible pigments of today, was to dissipate the incident solar light into heat. We have previously described the non-equilibrium thermodynamics and the photochemical mechanisms which may have been involved in the dissipative structuring of the purines adenine and hypoxanthine from the common precursor molecules of hydrogen cyanide and water under this UV light. In this article, we extend our analysis to include the production of the other two important purines, guanine and xanthine. The photochemical reactions are presumed to occur within a fatty acid vesicle floating on a hot (∼80 ∘C) neutral pH ocean surface exposed to the prevailing UV-C light. Reaction-diffusion equations are resolved under different environmental conditions. Significant amounts of adenine (∼10-5 M) and guanine (∼10-6 M) are obtained within 60 Archean days, starting from realistic concentrations of the precursors hydrogen cyanide and cyanogen (∼10-5 M).

Same modification, different location: the mythical role of N6-adenine methylation in plant genomes.

MAIN CONCLUSION: The present review summarizes recent advances in the understanding of 6mA in DNA as an emergent epigenetic mark with distinctive characteristics, discusses its importance in plant genomes, and highlights its chemical nature and functions. Adenine methylation is an epigenetic modification present in DNA (6mA) and RNA (m6A) that has a regulatory function in many cellular processes. This modification occurs through a reversible reaction that covalently binds a methyl group, usually at the N6 position of the purine ring. This modification carries biophysical properties that affect the stability of nucleic acids as well as their binding affinity with other molecules. DNA 6mA has been related to genome stability, gene expression, DNA replication, and repair mechanisms. Recent advances have shown that 6mA in plant genomes is related to development and stress response. In this review, we present recent advances in the understanding of 6mA in DNA as an emergent epigenetic mark with distinctive characteristics. We discuss the key elements of this modification, focusing mainly on its importance in plant genomes. Furthermore, we highlight its chemical nature and the regulatory effects that it exerts on gene expression and plant development. Finally, we emphasize the functions of 6mA in photosynthesis, stress, and flowering.

Nanoparticles Formulation Improves the Antifibrogenic Effect of Quercetin on an Adenine-Induced Model of Chronic Kidney Disease.

Renal fibrosis is the final stage of chronic kidney injury characterized by glomerulosclerosis and tubulointerstitial fibrosis with parenchymal destruction. Quercetin belongs to the most studied flavonoids with antioxidant, anti-inflammatory, antifibrogenic, and antitumor activity. It modifies the TGF-ß/Smad signaling pathway, decreasing profibrogenic expression molecules and inducing the expression of antioxidant, anti-inflammatory, and antifibrogenic molecules. However, quercetin exhibits poor water solubility and low absorption and bioavailability. This limitation was solved by developing a nanoparticles formulation that improves the solubility and bioavailability of several bioactive compounds. Therefore, we aimed to investigate the in vivo antifibrogenic effect of a quercetin nanoparticles formulation. Male C57BL/6 mice were induced into chronic renal failure with 50 mg/kg of adenine for four weeks. The animals were randomly grouped and treated with 25, 50, or 100 mg/kg of quercetin, either macroparticles or nanoparticles formulation. We performed biochemical, histological, and molecular analyses to evaluate and compare the effect of macroparticles versus nanoparticles formulation on kidney damage. Here, we demonstrated that smaller doses of nanoparticles exhibited the same beneficial effect as larger doses of macroparticles on preventing kidney damage. This finding translates into less quercetin consumption reaching the desired therapeutic effect.

Treatment of Chronic Kidney Disease with Extracellular Vesicles from Mesenchymal Stem Cells and CD133+ Expanded Cells: A Comparative Preclinical Analysis.

Chronic kidney disease (CKD) is characterized by structural abnormalities and the progressive loss of kidney function. Extracellular vesicles (EVs) from human umbilical cord tissue (hUCT)-derived mesenchymal stem cells (MSCs) and expanded human umbilical cord blood (hUCB)-derived CD133+ cells (eCD133+) maintain the characteristics of the parent cells, providing a new form of cell-free treatment. We evaluated the effects of EVs from hUCT-derived MSCs and hUCB-derived CD133+ cells on rats with CDK induced by an adenine-enriched diet. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The animals were randomized and divided into the MSC-EV group, eEPC-EV group and control group. Infusions occurred on the seventh and 14th days after CKD induction. Evaluations of kidney function were carried out by biochemical and histological analyses. Intense labeling of the α-SMA protein was observed when comparing the control with MSC-EVs. In both groups treated with EVs, a significant increase in serum albumin was observed, and the increase in cystatin C was inhibited. The results indicated improvements in renal function in CKD, demonstrating the therapeutic potential of EVs derived from MSCs and eCD133+ cells and suggesting the possibility that in the future, more than one type of EV will be used concurrently.

Enhanced resistance to Ca2+-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

The Dissipative Photochemical Origin of Life: UVC Abiogenesis of Adenine.

The non-equilibrium thermodynamics and the photochemical reaction mechanisms are described which may have been involved in the dissipative structuring, proliferation and complexation of the fundamental molecules of life from simpler and more common precursors under the UVC photon flux prevalent at the Earth's surface at the origin of life. Dissipative structuring of the fundamental molecules is evidenced by their strong and broad wavelength absorption bands in the UVC and rapid radiationless deexcitation. Proliferation arises from the auto- and cross-catalytic nature of the intermediate products. Inherent non-linearity gives rise to numerous stationary states permitting the system to evolve, on amplification of a fluctuation, towards concentration profiles providing generally greater photon dissipation through a thermodynamic selection of dissipative efficacy. An example is given of photochemical dissipative abiogenesis of adenine from the precursor HCN in water solvent within a fatty acid vesicle floating on a hot ocean surface and driven far from equilibrium by the incident UVC light. The kinetic equations for the photochemical reactions with diffusion are resolved under different environmental conditions and the results analyzed within the framework of non-linear Classical Irreversible Thermodynamic theory.

Raman and Fluorescence Profiles Modifications of Muscular and Adipose Tissues Exposed to Low Energy X-ray Beams.

This work aims to investigate changes induced by low-energy radiation in adipose and muscular tissues employing autofluorescence and Raman spectroscopic techniques. X-ray beams expositions with 25 and 35 kV at 0.11, 1.1, and 2.1 Gy radiation dose levels were applied. Changes in Raman line intensities at specific bands assigned to collagen, proteins, and lipids were observed. Autofluorescent analysis exhibit variations in the collagen and nicotinamide adenine dinucleotide emission (NADH), resulting from the structural modifications, variations on the reduced/oxidized fluorophores equilibrium followed by radiation exposure. Results show that Raman and fluorescence spectroscopy are suitable techniques to evaluate radiation effects on biomolecules even at low radiation doses and energies.

Adenine DNA methylation, 3D genome organization, and gene expression in the parasite Trichomonas vaginalis.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract causing infections that range from asymptomatic to highly inflammatory. Recent works have highlighted the importance of histone modifications in the regulation of transcription and parasite pathogenesis. However, the nature of DNA methylation in the parasite remains unexplored. Using a combination of immunological techniques and ultrahigh-performance liquid chromatography (UHPLC), we analyzed the abundance of DNA methylation in strains with differential pathogenicity demonstrating that N6-methyladenine (6mA), and not 5-methylcytosine (5mC), is the main DNA methylation mark in T. vaginalis Genome-wide distribution of 6mA reveals that this mark is enriched at intergenic regions, with a preference for certain superfamilies of DNA transposable elements. We show that 6mA in T. vaginalis is associated with silencing when present on genes. Interestingly, bioinformatics analysis revealed the presence of transcriptionally active or repressive intervals flanked by 6mA-enriched regions, and results from chromatin conformation capture (3C) experiments suggest these 6mA flanked regions are in close spatial proximity. These associations were disrupted when parasites were treated with the demethylation activator ascorbic acid. This finding revealed a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression in this divergent member of the Excavata.

Calculation of the Geometries and Infrared Spectra of the Stacked Cofactor Flavin Adenine Dinucleotide (FAD) as the Prerequisite for Studies of Light-Triggered Proton and Electron Transfer.

Flavin cofactors, like flavin adenine dinucleotide (FAD), are important electron shuttles in living systems. They catalyze a wide range of one- or two-electron redox reactions. Experimental investigations include UV-vis as well as infrared spectroscopy. FAD in aqueous solution exhibits a significantly shorter excited state lifetime than its analog, the flavin mononucleotide. This finding is explained by the presence of a "stacked" FAD conformation, in which isoalloxazine and adenine moieties form a π-complex. Stacking of the isoalloxazine and adenine rings should have an influence on the frequency of the vibrational modes. Density functional theory (DFT) studies of the closed form of FAD in microsolvation (explicit water) were used to reproduce the experimental infrared spectra, substantiating the prevalence of the stacked geometry of FAD in aqueous surroundings. It could be shown that the existence of the closed structure in FAD can be narrowed down to the presence of only a single water molecule between the third hydroxyl group (of the ribityl chain) and the N7 in the adenine ring of FAD.

Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice.

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.

Kinetic and oligomeric study of Leishmania braziliensis nicotinate/nicotinamide mononucleotide adenylyltransferase.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in REDOX reactions and oxidative stress defense systems. Furthermore, NAD is used as substrate by proteins that regulate essential cellular functions as DNA repair, genetic, and signal transduction, among many others. NAD biosynthesis can be completed through the de novo and salvage pathways, which converge at the common step catalyzed by the nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT EC: 2.7.7.1/18). Here, we report the kinetic characterization of the NMNAT of Leishmania braziliensis (LbNMNAT), one of the etiological agents of leishmaniasis, a relevant parasitic disease. The expression and homogeneous purification of the recombinant 6xHis-LbNMNAT protein was carried out and its kinetic study, which included analysis of K m , V max , K cat and the equilibrium constant (K D ) for both the forward and reverse reactions, was completed. The oligomeric state of the recombinant 6xHis-LbNMNAT protein was studied through size exclusion chromatography. Our results indicated the highest and lowest K m values for ATP and NAD, respectively. According to the calculated K D , the pyrophosphorolytic cleavage of NAD is favored in vitro. Moreover, the recombinant 6xHis-LbNMNAT protein showed a monomeric state, although it exhibits a structural element involved in potential subunits interaction. Altogether, our results denote notable differences of the LbNMNAT protein in relation to the human orthologs HsNMNAT1-3. These differences constitute initial findings that have to be continued to finally propose the NMNAT as a promissory pharmacological target in L. braziliensis.