The glutathione biosynthesis in the psychrophile Pseudoalteromonas haloplanktis

Reduced glutathione (GSH), together with its oxidized form (GSSG), is the most effective antioxidant system responsible for controlling the cellular redox state. Its biosynthesis from glutamate, cysteine and glycine, normally requires two enzymes. Indeed, γ-glutamyl-cysteine ligase (GshA) forms γ-glutamyl-cysteine, whereas glutathione synthetase (GshB) leads to the formation of GSH. In the genome of Pseudoalteromonas haloplanktis, a psychrophilic eubacterium isolated from Antarctic sea water, two genes coding for GshA (PhGshA-I and PhGshA-II ) and one gene for GshB (PhGshB) were putatively identified. The study of the biochemical properties of these enzymes was addressed with an appropriate heterologous expression system, thus leading to the production of the recombinant forms of PhGshB and PhGshA-II (rPhGshB and rPhGshA-II), purified by affinity chromatography. The first enzyme investigated was rPhGshB. Its purification was achieved either in the absence or in the presence of β-mercaptoethanol. The study of its molecular properties showed that, when purified in the presence of β-mercaptoethanol, rPhGshB underwent a covalent modification; however, this modification did not significantly affect its biochemical properties. The molecular mass of rPhGshB in denaturing conditions was 36 kDa, corresponding to the mass of the PhGshB monomer; in non denaturing conditions the mass determined by gel-filtration ranged between 74 and 136 kDa, respectively, values corresponding to a dimeric or tetrameric organization. The different behavior depended on the enzyme concentration and the data suggested that at higher concentrations the enzyme formed an unstable tetramer that at lower concentrations was converted into a dimeric and more stable form. To study the activity of rPhGshB, a new method for direct determination was developed, based on the hydrolysis of the radioactive substrate [γ32P] ATP; in fact, the synthesis of GSH catalyzed by GshB is coupled to the hydrolysis of ATP. The ATPase activity of rPhGshB required the presence of the other two substrates, glycine and γ-glutamylcysteine (γ-Glu-Cys). rPhGshB showed its maximum activity in the 7.4-8.2 pH range. The enzyme activity required also the presence of a divalent cation and at 5 mM Mg++ reached its maximum. The kinetic parameters of rPhGshB at 15°C, the optimum value for growth of P. haloplanktis, were determined. The enzyme showed a comparable affinity for ATP and γ-Glu-Cys (Km = 0.26 mM and 0.25 mM, respectively), whereas a lower affinity was determined for glycine (Km = 0.75 mM). The comparison of these data with those of the corresponding enzyme from other sources showed the remarkable similarity with Escherichia coli; a similar lower affinity for glycine compared to the other two substrates was found in the other sources. Enzyme inhibition studies showed that GSSG is an inhibitor of rPhGshB. The effect of temperature on the kinetic parameters of rPhGshB was analysed in the temperature range 10-30°C. The data showed that rPhGshB is already active at 10°C and its Vmax significantly increased with temperature up to 25°C; on the other hand, in the temperature interval considered, a minimum variation of Km was observed. The kcat values were then used to draw an Arrhenius plot and in the range of linearity (10-25°C) an activation energy of 75 kJ/mol was determined, a value quite high for a psychrophilic enzyme. The thermostability of rPhGshB was analyzed using a thermal inactivation profile and an extrapolated half-life of 10 min at 50.5°C was derived. This value was slightly high for a psychrophilic enzyme, although not unusual for enzymes involved in the control of the cellular redox state. A value of 208 kJ/mol was calculated for the energy of activation of the heat inactivation process; this value was intermediate between those usually obtained for psychrophilic and mesophilic enzymes, respectively. Finally, the development of a new crystallization technique allowed the obtainment of crystal forms of rPhGshB, useful for the determination of the three-dimensional structure of the enzyme by X-ray diffraction. The biochemical characterization of rPhGshA-II was started, using the same assay adopted for rPhGshB. Preliminary data obtained showed that the enzyme activity of rPhGshA-II requires a reducing agent in the reaction mixture; furthermore, the presence of the other two substrates glutamate and cysteine, is also required. The activity of rPhGshA-II needed a higher concentration of Mg++ (10-20 mM) compared to that already determined for rPhGshB. When the characterization of the molecular and biochemical properties of PhGshA-II will be completed, it will be possible to reconstitute in vitro for the first time the system for the biosynthesis of glutathione in a psychrophilic organism

Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P212121, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date

A review of the molecular mechanisms underlying the development and progression of cardiac remodeling

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems. Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively). Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, for the Ph.D grant of Catarina Silva (UMINHO/BD/33/2016; NORTE-08-5369-FSE-000012), and by the Portuguese Science and Technology Foundation (FCT) for the cells project Cells4_ID (PTDC/BTM-SAL/28882/2017)

SiRNA delivery for control of cyclin d1 and e2f1 expression in crohn’s disease

Evidence in inflammatory bowel diseases (IBD) supports a connection between inflammation and cancer due to the alteration of the cell cycle with loss of control at the G1/S checkpoint. In this study, we analyze the expression and modulation of CyD1 and E2F1 in colon explants from Crohn's disease (CD) patients. We used ex vivo culture of colon explants from 4 CD patients and 2 healthy controls, stimulated with lipopolysaccharide from Escherichia Coli (EC-LPS). Commercial siRNAs for CyD1 and E2F1 inhibition were encapsulated in Invivofectamine® and in purposely produced nanoliposomal vectors to silencing CyD1 and E2F1 expression. Western blot analysis was used to investigate the effect of siRNA on CyD1, E2F1 and cyclooxygenase 2 (COX-2) expression. In CD patients colon explants, CyD1 and E2F1 increased after the inflammatory stimulus but siRNA silencing attenuated their expression and controlled the COX-2 expression too. These data represent a prelimiary exploration of in vitro siRNA use

Unraveling the impact of upfront chemotherapy and proton beam therapy on treatment outcome and follow-up in central nervous system germ cell tumors: a single center experience

BackgroundGerm cell tumors (GCT) account for a minority of central nervous system (CNS) malignancies, highly prevalent in adolescents and young adults. Despite their aggressive biological behavior, prognosis is excellent in most cases with risk stratified treatment, consisting in a combination of chemotherapy and radiotherapy. Whole ventricular irradiation (WVI) and craniospinal irradiation, the treatment of choice for localized and metastatic disease, pose significant risk of collateral effects, therefore proton beam radiation (PBT) has been recently proposed for its steep dose fallout.Materials and methodsWe report our experience in a consecutive series of 17 patients treated for CNS GCT at our Institution from 2015 to 2021.ResultsMost frequent lesion location were sellar/suprasellar (35%) and bifocal germinoma (35%), followed by pineal (18%) and thalamic (12%). Two patients (12%), had evidence of disseminated disease at the time of diagnosis. At the latest follow-up all but one patient showed complete response to treatment. The only relapse was successfully rescued by additional chemotherapy and PBT. PBT was well tolerated in all cases. No visual, neurological or endocrinological worsening was documented during and after treatment. Neuropsychological evaluation demonstrated preservation of cognitive performance after PBT treatment.ConclusionsOur data, albeit preliminary, strongly support the favourable therapeutic profile of PBT for the treatment of CNS germ cell tumors

Analisi dei modelli organizzativi di risposta al COVID-19 in Italia: evidenze da 2 anni di ISTANT REPORT Altems

Sistematizzazione e pubblicazione settimanalmente un report per analizzare in modo sistemico e comparativo i modelli di risposta adottati dalle Regioni in risposta all'emergenza da COVID- 1

Proline and light as quality enhancers of rocket (Eruca sativa Miller) grown under saline conditions

Stress conditions experienced during growth may affect plant responses during post-harvest storage and eventually determine the overall quality of commercial products. In this context, we hypothesized that foliar applications of proline during the growth cycle and light exposure during post-harvest storage could be two important modulators of yield and quality parameters of rocket plants exposed to NaCl stress. Dry matter percentage increased upon NaCl treatment. However, fresh weight loss during storage did not change over time as a consequence of salt stress. High salinity (100 mM NaCl) moderately reduced both leaf nitrate (14%) and nitrite (3%) contents. Lipophilic (LAC) and hydrophilic (HAC) antioxidant activities also decreased by 10% at the highest salinization (average of two growth cycles). In contrast, during storage, LAC decreased whereas HAC increased. Proline applications reduced the leaf nitrate content during storage by 16%, increased carotenoids and chlorophyll contents in salinized plants and also increased the ascorbate leaf concentration in both salinized and non-salinzed plants. Light storage enhanced fresh weight loss in contrast to dark storage. However the exposure to light reduced leaf nitrate levels by 7% (average of two growth cycles) and contributed to maintain high leaf ascorbate concentrations over time. The combined control of plant physiological responses to environmental stressors and post-harvest storage parameters may affect the nutritional profile of fresh rocket and consequently should be considered to define standard production protocols to improve the nutritional qualities of commercial produce

Biochemical properties of the superoxide dismutase from the pathogenic bacterium Helicobacter pylori

Helicobacter pylori, a pathogenic aerotolerant bacterium colonizing the gastric mucosa, causes gastritis and ulcer and, if not eradicated, may lead to the development of gastric tumors. H. pylori resistance to moderate oxygen concentrations is mainly due to the key anti-oxidant role played by superoxide dismutase (HpSOD), the enzyme scavenging the toxic superoxide anions formed during oxygen consumption. The 3D structure of a recombinant form of HpSOD showed that this enzyme, belonging to the Fe-SOD family, contains an extended C–terminal tail, which is missing in other bacterial SODs and whose role remains obscure. Furthermore, in some cultures of H. pylori the endogenous HpSOD was found anchored to the flagellar sheath of the bacterium. In the present study the biochemical properties of the recombinant HpSOD were investigated to improve the knowledge on the enzyme functions, which could explain the H. pylori survival in the harsh conditions of the stomach. The research is also focused on the possible role played by the unusual C–terminal extension of HpSOD. The high specific activity (5000 U/mg) of the recombinant enzyme and its discrete heat resistance (T1/2 = 64C) ensure its functional efficiency. The effect of typical inhibitors and inactivators of SODs was investigated; while sodium azide caused only a low inhibition, hydrogen peroxide and peroxynitrite provoked a significant inactivation of HpSOD. It is known that the genes involved in glutathione biosynthesis are missing in the H. pylori genome; however, HpSOD undergoes a glutathionylation reaction by the oxidized form of glutathione. A mutagenic analysis aimed at the replacement of the two cysteine residues possessed by HpSOD allowed the identification of C79 as the target residue of the S–glutathionylation reaction. The production of a deleted form of HpSOD lacking the C–terminal extension of the enzyme is on the way, to check the effect on the biochemical properties of the enzyme

Método de construcción del plan de marketing con base en el modelo canvas para empresas incubadas

El objetivo del trabajo fue desarrollar un método que optimice la planificación de marketing para empresas incubadas. Para ello, como herramienta de estudio, se siguió el trabajo de cinco empresas incubadas en el CCENTEV. A través de las actividades realizadas y los estudios, se desarrollaron planes de marketing para las empresas y un modelo para la elaboración de un Plan de Marketing basado en el Método CANVAS, que reduce el tiempo, mano de obra, recursos humanos y financieros de una planificación, haciendo que el plan sea asequible para pequeñas y nuevas empresas. Para preparar el modelo CANVAS para marketing se seleccionaron ocho temas esenciales: público objetivo, objetivo, marca, marketing digital, análisis ambiental, medidas de control, marketing mix y relación con el cliente. Sin embargo, se sabe que las empresas tienen diferentes realidades y modos de operación en el mercado, por lo que se utilizó una segmentación según el grado de madurez de marketing de las organizaciones, con el objetivo de desarrollar un marco de marketing CANVAS para incluir diferentes tipos de empresas. Así, se aplicó el modelo en una empresa de nivel intermedio, y se obtuvieron los lineamientos de su plan de marketing de forma objetiva, práctica y fácil de entender. Se concluye que el modelo propuesto optimizó el tiempo de elaboración del plan de marketing y permitió a las nuevas empresas gestoras desarrollar su plan de marketing, relevante para la supervivencia de la organización en el mercado, asegurando ventajas competitivas y adaptación a las constantes evoluciones globales