Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

Crosstalk between P2Y receptors and cyclooxygenase activity in inflammation and tissue repair.

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.

Lipoxin-mediated signaling: ALX/FPR2 interaction and beyond.

In the aftermath of tissue injury or infection, an efficient resolution mechanism is crucial to allow tissue healing and preserve appropriate organ functioning. Pro-resolving bioactive lipids prevent uncontrolled inflammation and its consequences. Among these mediators, lipoxins were the first described and their pro-resolving actions have been mainly described in immune cells. They exert their actions mostly through formyl-peptide receptor 2 (ALX/FPR2 receptor), a G-protein-coupled receptor whose biological function is tremendously complex, primarily due to its capacity to mediate variable cellular responses. Moreover, lipoxins can also interact with alternative receptors like the cytoplasmic aryl hydrocarbon receptor, the cysteinyl-leukotrienes receptors or GPR32, triggering different intracellular signaling pathways. The available information about this complex response mediated by lipoxins is addressed in this review, going over the different mechanisms used by these molecules to stop the inflammatory reaction and avoid the development of dysregulated and chronic pathologies.

Combined Immune Defect in B-Cell Lymphoproliferative Disorders Is Associated with Severe Infection and Cancer Progression.

B cell chronic lymphoproliferative diseases (B-CLPD) are associated with secondary antibody deficiency and other innate and adaptive immune defects, whose impact on infectious risk has not been systematically addressed. We performed an immunological analysis of a cohort of 83 B-CLPD patients with recurrent and/or severe infections to ascertain the clinical relevance of the immune deficiency expression. B-cell defects were present in all patients. Patients with combined immune defect had a 3.69-fold higher risk for severe infection (p = 0.001) than those with predominantly antibody defect. Interestingly, by Kaplan-Meier analysis, combined immune defect showed an earlier progression of cancer with a hazard ratio of 3.21, than predominantly antibody defect (p = 0.005). When B-CLPD were classified in low-degree, high-degree, and plasma cell dyscrasias, risk of severe disease and cancer progression significantly diverged in combined immune defect, compared with predominantly antibody defect (p = 0.001). Remarkably, an underlying primary immunodeficiency (PID) was suspected in 12 patients (14%), due to prior history of infections, autoimmune and granulomatous conditions, atypical or variegated course and compatible biological data. This first proposed SID classification might have relevant clinical implications, in terms of predicting severe infections and cancer progression, and might be applied to different B-CLPD entities.

Specialized Proresolving Mediators Protect Against Experimental Autoimmune Myocarditis by Modulating Ca2+ Handling and NRF2 Activation.

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum-adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

The Aryl Hydrocarbon Receptor Ligand FICZ Improves Left Ventricular Remodeling and Cardiac Function at the Onset of Pressure Overload-Induced Heart Failure in Mice.

Adverse ventricular remodeling is the heart's response to damaging stimuli and is linked to heart failure and poor prognosis. Formyl-indolo [3,2-b] carbazole (FICZ) is an endogenous ligand for the aryl hydrocarbon receptor (AhR), through which it exerts pleiotropic effects including protection against inflammation, fibrosis, and oxidative stress. We evaluated the effect of AhR activation by FICZ on the adverse ventricular remodeling that occurs in the early phase of pressure overload in the murine heart induced by transverse aortic constriction (TAC). Cardiac structure and function were evaluated by cardiac magnetic resonance imaging (CMRI) before and 3 days after Sham or TAC surgery in mice treated with FICZ or with vehicle, and cardiac tissue was used for biochemical studies. CMRI analysis revealed that FICZ improved cardiac function and attenuated cardiac hypertrophy. These beneficial effects involved the inhibition of the hypertrophic calcineurin/NFAT pathway, transcriptional reduction in pro-fibrotic genes, and antioxidant effects mediated by the NRF2/NQO1 pathway. Overall, our findings provide new insight into the role of cardiac AhR signaling in the injured heart.

NOD1 splenic activation confers ferroptosis protection and reduces macrophage recruitment under pro-atherogenic conditions.

The bioavailability and regulation of iron is essential for central biological functions in mammals. The role of this element in ferroptosis and the dysregulation of its metabolism contribute to diseases, ranging from anemia to infections, alterations in the immune system, inflammation and atherosclerosis. In this sense, monocytes and macrophages modulate iron metabolism and splenic function, while at the same time they can worsen the atherosclerotic process in pathological conditions. Since the nucleotide-binding oligomerization domain 1 (NOD1) has been linked to numerous disorders, including inflammatory and cardiovascular diseases, we investigated its role in iron homeostasis. The iron content was measured in various tissues of Apoe-/- and Apoe-/-Nod1-/- mice fed a high-fat diet (HFD) for 4 weeks, under normal or reduced splenic function after ligation of the splenic artery. In the absence of NOD1 the iron levels decreased in spleen, heart and liver regardless the splenic function. This iron decrease was accompanied by an increase in the recruitment of F4/80+-macrophages in the spleen through a CXCR2-dependent signaling, as deduced by the reduced recruitment after administration of a CXCR2 inhibitor. CXCR2 mediates monocyte/macrophage chemotaxis to areas of inflammation and accumulation of leukocytes in the atherosclerotic plaque. Moreover, in the absence of NOD1, inhibition of CXCR2 enhanced atheroma progression. NOD1 activation increased the levels of GPX4 and other iron and ferroptosis regulatory proteins in macrophages. Our findings highlight the preeminent role of NOD1 in iron homeostasis and ferroptosis. These results suggest promising avenues of investigation for the diagnosis and treatment of iron-related diseases directed by NOD1.

The Informal Social Support for Autonomy and Dependence in Pain Inventory Spanish version.

Social support plays a crucial role in the quality of life of people with chronic pain. The Informal Social Support for Autonomy and Dependence in Pain Inventory assesses two functions of received social support: the promotion of autonomy and the promotion of dependence. The aim of this cross-sectional study was to adapt this instrument for its use in the Spanish population. The sample comprised 256 individuals with chronic pain. Participants were recruited through two local associations of people with fibromyalgia, a physiotherapy unit and a hospital pain unit. The data were collected in Spain between October 2018 and January 2020. The structure of the questionnaire was analysed using confirmatory factor analysis, average variance extracted, composite reliability and internal consistency indexes, and inter-correlations between the scales. The criterion-related validity of the instrument was analysed by investigating its relationship with pain intensity, positive and negative affect, daily functioning, activity impairment, wellbeing and satisfaction with life. The structure with the best fit had four related factors: emotional social support for the promotion of autonomy; instrumental social support for the promotion of autonomy; emotional social support for the promotion of dependence and instrumental social support for the promotion of dependence. The scales showed adequate internal consistency. An association was found between higher levels of instrumental social support for the promotion of dependence and higher levels of pain-related disability and decreased daily functioning. An association was also found between the promotion of autonomy and increased satisfaction with life. The Spanish version of the inventory shows appropriate psychometric properties. In the setting of disability prevention, this instrument is useful in assessing the support relationships between people with chronic pain and their relatives.

Beneficial effects of paricalcitol on cardiac dysfunction and remodelling in a model of established heart failure.

BACKGROUND AND PURPOSE: The synthetic vitamin D3 analogue paricalcitol acts as a selective activator of the vitamin D receptor (VDR). While there is evidence for cardioprotective effects of paricalcitol associated with the VDR pathway, less information is available about the structural and functional cardiac effects of paricalcitol on established heart failure (HF) and particularly its effects on associated electrophysiological or Ca2+ handling remodelling. EXPERIMENTAL APPROACH: We used a murine model of transverse aortic constriction (TAC) to study the effect of paricalcitol on established HF. Treatment was initiated 4 weeks after surgery over five consecutive weeks, and mice were sacrificed 9 weeks after surgery. Cardiac MRI (CMRI) was performed 4 and 9 weeks after surgery. Hearts were used for biochemical and histological studies and to isolate ventricular myocytes for electrophysiological and calcium imaging studies. KEY RESULTS: CMRI analysis revealed that, compared with vehicle, paricalcitol treatment prevented the progression of ventricular dilation and hypertrophy after TAC and halted the corresponding decline in ejection fraction. These beneficial effects were related to the attenuation of intracellular Ca2+ mishandling remodelling, antifibrotic and antihypertrophic effects and potentially antiarrhythmic effects by preventing the reduction of K+ current density and the long QT, JT and TpTe intervals observed in HF animals. CONCLUSION AND IMPLICATIONS: The results suggest that paricalcitol treatment in established HF hampers disease progression and improves adverse electrophysiological and Ca2+ handling remodelling, attenuating the vulnerability to HF-associated ventricular arrhythmias. Paricalcitol may emerge as a potential therapeutic option in the treatment of HF.

Hipertensión arterial pulmonar:: progresión, pronóstico e impacto

La hipertensión arterial pulmonar es una enfermedad infrecuente, caracterizada por el aumento de postcarga del ventrículo derecho. La adaptación del ventrículo derecho a esta postcarga se relaciona con el pronóstico de los pacientes. La estratificación pronóstica se basa en una valoración multiparamétrica. Los parámetros clínicos, funcionales, la valoración del ventrículo derecho y la evaluación hemodinámica van a definir la situación de riesgo de los pacientes, en función de la mortalidad a un año. La situación de bajo riesgo a un año de seguimiento se ha relacionado con mayor supervivencia. El algoritmo terapéutico actual incluye como estrategia inicial la mono, doble y triple terapia. Se debe realizar escalada terapéutica con el objetivo de mantener a los pacientes en bajo riesgo durante el seguimiento.

Pulmonary arterial hypertension is an infrequent disease, characterized by an increase in afterload of the right ventricle. The adaptation of the right ventricle to this afterload is related to the prognosis of the patients. The prognostic stratification is based on a multiparametric assessment. The clinical, functional parameters, assessment of the right ventricle and hemodynamic evaluation will define the risk situation of the patients, depending on the one-year mortality. The situation of low risk at one year of follow-up has been related to greater survival. The current therapeutic algorithm includes monotherapy, double and triple therapy as an initial strategy. Therapeutic escalation should be carried out in order to keep patients at low risk during follow-up.

A hipertensão arterial pulmonar é uma doença infreqüente, caracterizada por aumento da pós-carga do ventrículo direito. A adaptação do ventrículo direito a essa pós-carga está relacionada ao prognóstico dos pacientes. A estratificação prognóstica é baseada em uma avaliação multiparamétrica. Os parâmetros clínicos, funcionais, avaliação do ventrículo direito e avaliação hemodinâmica definirão a situação de risco dos pacientes, dependendo da mortalidade em um ano. A situação de baixo risco em um ano de seguimento tem sido relacionada à maior sobrevida. O algoritmo terapêutico atual inclui como estratégia inicial a monoterapia e a terapia dupla e tripla. O escalonamento terapêutico deve ser realizado a fim de manter os pacientes em baixo risco durante o acompanhamento.

Gender-Dependent Alteration of Ca2+ and TNFα Signaling in db/db Mice, an Obesity-Linked Type 2 Diabetic Model.

Cardiovascular complications are the primary death cause in type 2 diabetes, where inflammation can play a role. We, and others, have previously shown that, in diabetic cardiomyopathy, cardiac dysfunction is associated with Ca2+ mishandling. It is possible that diabetic cardiomyopathy differently affects men and women, as the latter present higher risk to develop heart failure and a higher plasmatic level of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα), than men. However, the gender-dependent regulation of Ca2+ signaling in diabetes and its relationship with TNFα signaling are still unclear. Here, we analyzed TNFα signaling pathway and its role in Ca2+ signaling dysfunction in male and female rodent models of type 2 diabetes linked to obesity (db/db mice) using confocal microscopy in freshly isolated cardiomyocytes. TNFα increased [Ca2+]i transient amplitude and accelerated its decay without affecting SR Ca2+ load or Ca2+ spark frequency in cells from control mice. All TNFα effects on Ca2+ handling were prevented by the inhibition of the ceramidase and the phospholipase A2 (PLA2). While the plasmatic level of TNFα was similar in male and female db/db mice, only male db/db hearts over-expressed both TNFα converting enzyme (TACE) and the protective TNFα receptors 2 (TNF-R2). TNFα receptor 1 (TNF-R1) expression, involved in negative inotropic response of TNFα, was unchanged in both male and female db/db mice compared to controls. We found that male db/db mice cardiomyocytes presented a decrease in [Ca2+]i transient amplitude associated to a drop of sarcoplasmic reticulum Ca2+ load, not seen in female db/db mice. Interestingly, sustained incubation with TNFα did not restored Ca2+ signaling alteration observed in male db/db mice but still induces an increase in Ca2+ spark frequency as seen in control littermates. In cardiomyocytes from female db/db mice, TNFα had no visible effects on Ca2+ handling. In conclusion, our study shows that the alteration of Ca2+ signaling and TNFα, seen in db/db mice, is gender specific presenting an increase in TNFα cardio-protective pathway in male mice.

Fibroblast growth factor-23 promotes rhythm alterations and contractile dysfunction in adult ventricular cardiomyocytes.

BACKGROUND: Cardiac dysfunction and arrhythmia are common and onerous cardiovascular events in end-stage renal disease (ESRD) patients, especially those on dialysis. Fibroblast growth factor (FGF)-23 is a phosphate-regulating hormone whose levels dramatically increase as renal function declines. Beyond its role in phosphorus homeostasis, FGF-23 may elicit a direct effect on the heart. Whether FGF-23 modulates ventricular cardiac rhythm is unknown, prompting us to study its role on excitation-contraction (EC) coupling. METHODS: We examined FGF-23 in vitro actions on EC coupling in adult rat native ventricular cardiomyocytes using patch clamp and confocal microscopy and in vivo actions on cardiac rhythm using electrocardiogram. RESULTS: Compared with vehicle treatment, FGF-23 induced a significant decrease in rat cardiomyocyte contraction, L-type Ca2+ current, systolic Ca2+ transients and sarcoplasmic reticulum (SR) load and SR Ca2+-adenosine triphosphatase 2a pump activity. FGF-23 induced pro-arrhythmogenic activity in vitro and in vivo as automatic cardiomyocyte extracontractions and premature ventricular contractions. Diastolic spontaneous Ca2+ leak (sparks and waves) was significantly increased by FGF-23 via the calmodulin kinase type II (CaMKII)-dependent pathway related to hyperphosphorylation of ryanodine receptors at the CaMKII site Ser2814. Both contraction dysfunction and spontaneous pro-arrhythmic Ca2+ events induced by FGF-23 were blocked by soluble Klotho (sKlotho). CONCLUSIONS: Our results show that FGF-23 reduces contractility and enhances arrhythmogenicity through intracellular Ca2+ mishandling. Blocking its actions on the heart by improving sKlotho bioavailability may enhance cardiac function and reduce arrhythmic events frequently observed in ESRD.

Beneficial effects of leptin treatment in a setting of cardiac dysfunction induced by transverse aortic constriction in mouse.

KEY POINTS: Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart. Controversy exists regarding the adverse and beneficial effects of leptin on the heart We analysed the effect of a non-hypertensive dose of leptin on cardiac function, [Ca2+ ]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure-overload by transverse aorta constriction. We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype. These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca2+ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca2+ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca2+ handling parameters. ABSTRACT: Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca2+ ]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure-overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg-1  day-1 ) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca2+ ]i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin-treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca2+ waves. These proarrhythmic manifestations, related to Ca2+ /calmodulin-dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level.

Role of NOD1 in Heart Failure Progression via Regulation of Ca2+ Handling.

BACKGROUND: Heart failure (HF) is a complex syndrome associated with a maladaptive innate immune system response that leads to deleterious cardiac remodeling. However, the underlying mechanisms of this syndrome are poorly understood. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a newly recognized innate immune sensor involved in cardiovascular diseases. OBJECTIVES: This study evaluated the role of NOD1 in HF progression. METHODS: NOD1 was examined in human failing myocardium and in a post-myocardial infarction (PMI) HF model evaluated in wild-type (wt-PMI) and Nod1-/- mice (Nod1-/--PMI). RESULTS: The NOD1 pathway was up-regulated in human and murine failing myocardia. Compared with wt-PMI, hearts from Nod1-/--PMI mice had better cardiac function and attenuated structural remodeling. Ameliorated cardiac function in Nod1-/--PMI mice was associated with prevention of Ca2+ dynamic impairment linked to HF, including smaller and longer intracellular Ca2+ concentration transients and a lesser sarcoplasmic reticulum Ca2+ load due to a down-regulation of the sarcoplasmic reticulum Ca2+-adenosine triphosphatase pump and by augmented levels of the Na+/Ca2+ exchanger. Increased diastolic Ca2+ release in wt-PMI cardiomyocytes was related to hyperphosphorylation of ryanodine receptors, which was blunted in Nod1-/--PMI cardiomyocytes. Pharmacological blockade of NOD1 also prevented Ca2+ mishandling in wt-PMI mice. Nod1-/--PMI mice showed significantly fewer ventricular arrhythmias and lower mortality after isoproterenol administration. These effects were associated with lower aberrant systolic Ca2+ release and with a prevention of the hyperphosphorylation of ryanodine receptors under isoproterenol administration in Nod1-/--PMI mice. CONCLUSIONS: NOD1 modulated intracellular Ca2+ mishandling in HF, emerging as a new target for HF therapy.

Calcitriol (1,25-dihydroxyvitamin D3) increases L-type calcium current via protein kinase A signaling and modulates calcium cycling and contractility in isolated mouse ventricular myocytes.

BACKGROUND: Calcitriol, the bioactive metabolite of vitamin D, exerts its effects through interaction with the nuclear vitamin D receptor (VDR) to induce genomic responses. Calcitriol may also induce rapid responses via plasma membrane-associated VDR, involving the activation of second messengers and modulation of voltage-dependent channels. VDR is expressed in cardiomyocytes, but the molecular and cellular mechanisms involved in the rapid responses of calcitriol in the heart are poorly understood. OBJECTIVE: The aim of the present study was to analyze the rapid nongenomic effect of calcitriol on L-type calcium channels, intracellular Ca2+ ([Ca2+]i) transients, and cell contractility in ventricular myocytes. METHODS: We used the whole-cell patch-clamp technique to record L-type calcium current (ICaL) and confocal microscopy to study global [Ca2+]i transients evoked by electrical stimulation and cell shortening in adult mouse ventricular myocytes treated with vehicle or with calcitriol. In some experiments, ICaL was recorded using the perforated patch-clamp technique. RESULTS: Calcitriol treatment of cardiomyocytes induced a concentration-dependent increase in ICaL density (Half maximal effective concentration (EC50) = 0.23 nM) and a significant increase in peak [Ca2+]i transients and cell contraction. The effect of calcitriol on ICaL was prevented by pretreatment of cardiomyocytes with the protein kinase A (PKA) inhibitor KT-5720 but not with the ß-adrenergic blocker propranolol. The effect of calcitriol on ICaL was absent in myocytes isolated from VDR knockout mice. CONCLUSION: Calcitriol induces a rapid response in mouse ventricular myocytes that involves a VDR-PKA-dependent increase in ICaL density, enhancing [Ca2+]i transients and contraction.

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase.

It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+) accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+) when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4(+). Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+) when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.

Study of the putative fusion regions of the preS domain of hepatitis B virus.

In a previous study, it was shown that purified preS domains of hepatitis B virus (HBV) could interact with acidic phospholipid vesicles and induce aggregation, lipid mixing and leakage of internal contents which could be indicative of their involvement in the fusion of the viral and cellular membranes (Núñez, E. et al. 2009. Interaction of preS domains of hepatitis B virus with phospholipid vesicles. Biochim. Biophys. Acta 17884:417-424). In order to locate the region responsible for the fusogenic properties of preS, five mutant proteins have been obtained from the preS1 domain of HBV, in which 40 amino acids have been deleted from the sequence, with the starting point of each deletion moving 20 residues along the sequence. These proteins have been characterized by fluorescence and circular dichroism spectroscopy, establishing that, in all cases, they retain their mostly non-ordered conformation with a high percentage of ß structure typical of the full-length protein. All the mutants can insert into the lipid matrix of dimyristoylphosphatidylglycerol vesicles. Moreover, we have studied the interaction of the proteins with acidic phospholipid vesicles and each one produces, to a greater or lesser extent, the effects of destabilizing vesicles observed with the full-length preS domain. The ability of all mutants, which cover the complete sequence of preS1, to destabilize the phospholipid bilayers points to a three-dimensional structure and/or distribution of amino acids rather than to a particular amino acid sequence as being responsible for the membrane fusion process.

NOD1 receptor is up-regulated in diabetic human and murine myocardium.

Type 2 diabetes has a complex pathology that involves a chronic inflammatory state. Emerging evidence suggests a link between the innate immune system receptor NOD1 (nucleotide-binding and oligomerization domain 1) and the pathogenesis of diabetes, in monocytes and hepatic and adipose tissues. The aim of the present study was to assess the role of NOD1 in the progression of diabetic cardiomyopathy. We have measured NOD1 protein in cardiac tissue from Type 2 diabetic (db) mice. Heart and isolated cardiomyocytes from db mice revealed a significant increase in NOD1, together with an up-regulation of nuclear factor κB (NF-κB) and increased apoptosis. Heart tissue also exhibited an enhanced expression of pro-inflammatory cytokines. Selective NOD1 activation with C12-γ-D-glutamyl-m-diaminopimelic acid (iEDAP) resulted in an increased NF-κB activation and apoptosis, demonstrating the involvement of NOD1 both in wild-type and db mice. Moreover, HL-1 cardiomyocytes exposed to elevated concentrations of glucose plus palmitate displayed an enhanced NF-κB activity and apoptotic profile, which was prevented by silencing of NOD1 expression. To address this issue in human pathology, NOD1 expression was evaluated in myocardium obtained from patients with Type 2 diabetes (T2DMH) and from normoglycaemic individuals without cardiovascular histories (NH). We have found that NOD1 was expressed in both NH and T2DMH; however, NOD1 expression was significantly pronounced in T2DMH. Furthermore, both the pro-inflammatory cytokine tumour necrosis factor α (TNF-α) and the apoptosis mediator caspase-3 were up-regulated in T2DMH samples. Taken together, our results define an active role for NOD1 in the heightened inflammatory environment associated with both experimental and human diabetic cardiac disease.

Prolonged leptin treatment increases transient outward K⁺ current via upregulation of Kv4.2 and Kv4.3 channel subunits in adult rat ventricular myocytes.

Circulating leptin levels are elevated in obesity and hyperleptinaemia has been postulated to be an independent risk factor for the development of cardiovascular diseases. Although many studies have been published on the mechanisms involved in the effects of leptin on cardiac function and pathological remodeling, scarce information is currently available analyzing the influence of prolonged leptin treatment on ionic cardiac channels remodeling in adult ventricular myocytes. Enzymatically isolated adult rat ventricular myocytes were treated with leptin or vehicle for 48h. Real-Time RT-PCR were used to analyze mRNA expression of Kir2.1, Cav1.2, Cav 3.1, Kv4.2 and Kv4.3 α-subunits and KChIP2 auxiliary subunit. The fast transient outward potassium channels (Itof) α-subunits Kv4.2, Kv4.3 and KChIP2 were analyzed by Western-blot. The fast transient outward potassium current and the action potentials were recorded in isolated myocytes by the whole-cell patch-clamp technique. Leptin treatment induced an up-regulation of Kv4.2, Kv4.3 and KChIP2 subunits mRNA expression. However, transcriptional levels of Kir2.1, Cav1.2, or Cav3.1 α-subunit channels were unmodified by leptin. Protein expression levels of Kv4.2, Kv4.3 and KChIP2 subunits were also increased by leptin. The electrophysiological study showed that leptin increases the fast transient outward potassium current amplitudes and densities shortening action potential duration. In addition, leptin activated Akt signaling in cardiomyocytes and this mechanism was involved in the effect of leptin on Itof channels. In conclusión, leptin increases both the expression and function of Itof channels in adult ventricular myocytes and this mechanism involves Akt signaling. Altogether these data suggest that leptin could exert beneficial or detrimental effects depending on the initial ventricular myocyte repolarizing reserve.

Spectroscopic characterization and fusogenic properties of preS domains of duck hepatitis B virus.

In order to shed light on the hepatitis B virus fusion mechanism and to explore the fusogenic capabilities of preS regions, a recombinant duck hepatitis B virus (DHBV) preS protein (DpreS) containing six histidines at the carboxy-terminal end has been obtained. The DpreS domain, which has an open and mostly nonordered conformation as indicated by fluorescence and circular dichroism spectroscopies, has the ability to interact with negatively charged phospholipid vesicles. The observed interaction differences between neutral and acidic phospholipids can be interpreted in terms of an initial ionic interaction between the phospholipid polar headgroup and the protein followed by the insertion of probably the N-terminal region in the cellular membrane. Fluorescence polarization studies detect a decrease of the transition enthalpy together with a small modification of the transition temperature, typical effects of integral membrane proteins. The interaction of the protein with acidic phospholipid vesicles induces aggregation, lipid mixing, and leakage of internal contents, properties that have been ascribed to membrane destabilizing proteins. The fact that the preS domains of the hepadnaviruses have little similarity but share a very similar hydrophobic profile points to the importance of the overall three-dimensional structure as well as to its conformational flexibility and the distribution of polar and apolar amino acids on the expression of their destabilizing properties rather than to a particular amino acid sequence. The results presented herein argue for the involvement of DpreS in the initial steps of DHBV infection. Taken together with previously reported results, the conclusion that both S and preS regions participate in the fusion process of the hepadnaviridae family may be drawn.