Activation of cannabinoid type 1 receptor (CB1) modulates oligodendroglial process branching complexity in rat hippocampal cultures stimulated by olfactory ensheathing glia-conditioned medium.

The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.

Copper regulation disturbance linked to oxidative stress and cell death during Zika virus infection in human astrocytes.

The Zika virus (ZIKV) caused neurological abnormalities in more than 3500 Brazilian newborns between 2015 and 2020. Data have pointed to oxidative stress in astrocytes as well as to dysregulations in neural cell proliferation and cell cycle as important events accounting for the cell death and neurological complications observed in Congenital Zika Syndrome. Copper imbalance has been shown to induce similar alterations in other pathologies, and disturbances in copper homeostasis have already been described in viral infections. Here, we investigated copper homeostasis imbalance as a factor that could contribute to the cytotoxic effects of ZIKV infection in astrocytes. Human induced pluripotent stem cell-derived astrocytes were infected with ZIKV; changes in the gene expression of copper homeostasis proteins were analyzed. The effect of the administration of CuCl2 or a copper chelator on oxidative stress, cell viability and percentage of infection were also studied. ZIKV infection leads to a downregulation of one of the transporters mediating copper release, ATP7B protein. We also observed the activation of mechanisms that counteract high copper levels, including the synthesis of copper chaperones and the reduction of the copper importer protein CTR1. Finally, we show that chelator-mediated copper sequestration in ZIKV-infected astrocytes reduces the levels of reactive oxygen species and improves cell viability, but does not change the overall percentage of infected cells. In summary, our results show that copper homeostasis imbalance plays a role in the pathology of ZIKV in astrocytes, indicating that it may also be a factor accounting for the developmental abnormalities in the central nervous system following viral infection. Evaluating micronutrient levels and the use of copper chelators in pregnant women susceptible to ZIKV infection may be promising strategies to manage novel cases of congenital ZIKV syndrome.

Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way.

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na+ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na+ + K+-ATPase and the ouabain-insensitive, K+-insensitive, and furosemide-sensitive Na+-ATPase, which participate in the active Na+ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na+-ATPase activity (C1P50 ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na+-ATPase. Conversely, we did not observe any effect on the Na+ + K+-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na+-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Microglial lysophosphatidic acid promotes glioblastoma proliferation and migration via LPA1 receptor.

Glioblastomas (GBMs) are highly aggressive primary brain tumors characterized by cellular heterogeneity, insensitivity to chemotherapy and poor patient survival. Lysophosphatidic acid (LPA) is a lysophospholipid that acts as a bioactive signaling molecule and plays important roles in diverse biological events during development and disease, including several cancer types. Microglial cells, the resident macrophages of the central nervous system, express high levels of Autotaxin (ATX,Enpp2), an enzyme that synthetizes LPA. Our study aimed to investigate the role of LPA on tumor growth and invasion in the context of microglia-GBM interaction. First, through bioinformatics studies, patient data analysis demonstrated that more aggressive GBM expressed higher levels of ENPP2, which was also associated with worse patient prognosis with proneural GBM. Using GBM-microglia co-culture system we then demonstrated that GBM secreted factors were able to increase LPA1 and ATX in microglia, which could be further enhanced by hypoxia. On the other hand, interaction with microglial cells also increased ATX expression in GBM. Furthermore, microglial-induced GBM proliferation and migration could be inhibited by pharmacological inhibition of LPA1 , suggesting that microglial-derived LPA could support tumor growth and invasion. Finally, increased LPA1 expression was observed in GBM comparing with other gliomas and could be also associated with worse patient survival. These results show for the first time a microglia-GBM interaction through the LPA pathway with relevant implications for tumor progression. A better understanding of this interaction can lead to the development of new therapeutic strategies setting LPA as a potential target for GBM treatment.

Fluorescence cross-correlation spectroscopy as a valuable tool to characterize cationic liposome-DNA nanoparticle assembly.

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17.

BACKGROUND: Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another. OBJECTIVES: This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS: The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-ß-cyclodextrin (MßCD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy. FINDINGS: In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells. CONCLUSIONS: The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17

BACKGROUND Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another. OBJECTIVES This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-β-cyclodextrin (MβCD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy. FINDINGS In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells. CONCLUSIONS The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

Author Correction: Specific Phospholipids Regulate the Acquisition of Neuronal and Astroglial Identities in Post-Mitotic Cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

L-Tyr-Induced Phosphorylation of Tyrosine Hydroxylase at Ser40: An Alternative Route for Dopamine Synthesis and Modulation of Na+/K+-ATPase in Kidney Cells.

BACKGROUND/AIMS: Dopamine (DA) is a natriuretic hormone that inhibits renal sodium reabsorption, being Angiotensin II (Ang II) its powerful counterpart. These two systems work together to maintain sodium homeostasis and consequently, the blood pressure (BP) within normal limits. We hypothesized that L-tyrosine (L-tyr) or L-dihydroxyphenylalanine (L-dopa) could inhibit the Na+/K+-ATPase activity. We also evaluated whether L-tyr treatment modulates Tyrosine Hydroxylase (TH). METHODS: Experiments involved cultured LLCPK1 cells treated with L-tyr or L-dopa for 30 minutes a 37°C. In experiments on the effect of Dopa Descarboxylase (DDC) inhibition, cells were pre incubated for 15 minutes with 3-Hydroxybenzylhydrazine dihydrochloride (HBH), and them L-dopa was added for 30 minutes. Na+/K+-ATPase activity was quantified colorimetrically. We used immunoblotting and immunocytochemistry to identify the enzymes TH, DDC and the dopamine receptor D1R in LLCPK1 cells. TH activity was accessed by immunoblotting (increase in the phosphorylation). TH and DDC activities were also evaluated by the modulation of the Na+/K+-ATPase activity, which can be ascribed to the synthesis of dopamine. RESULTS: LLCPK1 cells express the required machinery for DA synthesis: the enzymes TH, and (DDC) as well as its receptor D1R, were detected in control steady state cells. Cells treated with L-tyr or L-dopa showed an inhibition of the basolateral Na+/K+-ATPase activity. We can assume that DA formed in the cytoplasm from L-tyr or L-dopa led to inhibition of the Na+/K+-ATPase activity compared to control. L-tyr treatment increases TH phosphorylation at Ser40 by 100%. HBH, a specific DDC inhibitor; BCH, a LAT2 inhibitor; and Sch 23397, a specific D1R antagonist, totally suppressed the inhibition of Na+/K+-ATPase activity due to L-dopa or L-tyr administration, as indicated in the figures. CONCLUSION: The results indicate that DA formed mainly from luminal L-tyr or L-dopa uptake by LAT2, can inhibit the Na+/K+-ATPase. In addition, our results showed for the very first time that TH activity is also significantly increased when the cells were exposed to L-tyr.

Experimental ischemia/reperfusion model impairs endocannabinoid signaling and Na+/K+ ATPase expression and activity in kidney proximal tubule cells.

LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, were upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner in the LLC-PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down-regulated following IR in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.

Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System.

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.

Specific Phospholipids Regulate the Acquisition of Neuronal and Astroglial Identities in Post-Mitotic Cells.

Hitherto, the known mechanisms underpinning cell-fate specification act on neural progenitors, affecting their commitment to generate neuron or glial cells. Here, we show that particular phospholipids supplemented in the culture media modify the commitment of post-mitotic neural cells in vitro. Phosphatidylcholine (PtdCho)-enriched media enhances neuronal differentiation at the expense of astroglial and unspecified cells. Conversely, phosphatidylethanolamine (PtdEtn) enhances astroglial differentiation and accelerates astrocyte maturation. The ability of phospholipids to modify the fate of post-mitotic cells depends on its presence during a narrow time-window during cell differentiation and it is mediated by the selective activation of particular signaling pathways. While PtdCho-mediated effect on neuronal differentiation depends on cAMP-dependent kinase (PKA)/calcium responsive element binding protein (CREB), PtdEtn stimulates astrogliogenesis through the activation of the MEK/ERK signaling pathway. Collectively, our results provide an additional degree of plasticity in neural cell specification and further support the notion that cell differentiation is a reversible phenomenon. They also contribute to our understanding of neuronal and glial lineage specification in the central nervous system, opening up new avenues to retrieve neurogenic capacity in the brain.

Long-term effect of a chronic low-protein multideficient diet on the heart: Hypertension and heart failure in chronically malnourished young adult rats.

BACKGROUND: We investigated whether a chronic low-protein multideficient diet (BRD) from weaning turns on cardiovascular adaptive responses that could culminate in hypertension and heart failure. METHODS AND RESULTS: Systolic pressure (SP) and heart rate (HR) were determined in CTRL (normal diet) and BRD rats. Plasma albumin, plasma urea and urinary urea excretion decreased in BRD rats. In this group, echocardiography and the Langendorff technique showed: (i) increased HR and hypertension; (ii) decreased LVDP, dP/dtmax, dP/dtmin, cardiac output, ejection fraction, stroke volume and left ventricular diameter. BRD rats were less sensitive to isoproterenol (ISO) in LVDP and dP/dtmax, with unchanged dP/dtmin; Pressure-volume relationships indicated left-oriented shifts in LVDP, SP and DP, and decreased capacitance compared to CTRL. BRD rats had higher cardiac and lung indexes, accompanied by muscle atrophy and recent ventricular-infarcted areas, higher ventricular ß1-AR content, and decreased ß2-AR and α1-AR. Propranolol treatment gave similar ISO responses in both groups, disappearance of the infarcted regions and, except for ß2-AR, recovery of normal receptor expression. BRD rats had intense stimulation of plasma membrane Ca2+-ATPase (PMCA) activity, with increased Ca2+ affinity and inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Ventricular phospholamban increased and Na+/Ca2+ exchanger decreased. PMCA activity correlated with an increase in its PKC-mediated phosphorylation, overlying a decrease in PKA-catalyzed phosphorylation. Propranolol normalized PKC and PKA activities with recovery of PMCA but not SERCA. CONCLUSION: BRD triggers sympathetic exacerbation and dysfunction in Ca2+ handling, accompanied by early onset of hypertension and left ventricle congestive heart failure.

Transplantation of bone marrow-derived MSCs improves renal function and Na++K+-ATPase activity in rats with renovascular hypertension.

Renovascular hypertension (RVH) is a progressive disease, leading to chronic kidney disease when untreated and no specific treatment is available. Therefore, development of new therapeutic modalities is imperative. RVH is triggered by renal artery stenosis and subsequent renin-angiotensin-aldosterone system activation; it can be experimentally induced by the 2 Kidneys-1 Clip (2K1C) model. This study investigates the therapeutic potential of renal subcapsular mesenchymal stem cell (MSC) infusion in 2K1C rats. Renal morphological and functional changes were analyzed, including Na++K+-ATPase activity and expression, renin angiotensin-converting enzyme (ACE) and angiotensin-II type 1 (AT1R) and type 2 (AT2R) receptors expression. 2K1C rats developed hypertension accompanied by renin upregulation (clipped kidney) and renal Na++K+-ATPase activity and expression reduction. MSC therapy decreased systolic blood pressure, renin, ACE, and AT1R, upregulated AT2R and podocin expression and restored renal Na++K+-ATPase activity and expression. In addition, MSC improved renal morphology, reduced fibrosis and TGF-ß expression in the clipped kidney, decreased proteinuria and restored protein plasma levels. In conclusion, transplantation into a renal subcapsule is an efficient route and MSC is a good candidate for cell therapy, which may represent an interesting approach for chronic kidney disease treatment.

Respiratory and Systemic Effects of LASSBio596 Plus Surfactant in Experimental Acute Respiratory Distress Syndrome.

BACKGROUND/AIMS: Exogenous surfactant has been proposed as adjunctive therapy for acute respiratory distress syndrome (ARDS), but it is inactivated by different factors present in the alveolar space. We hypothesized that co-administration of LASSBio596, a molecule with significant anti-inflammatory properties, and exogenous surfactant could reduce lung inflammation, thus enabling the surfactant to reduce edema and improve lung function, in experimental ARDS. METHODS: ARDS was induced by cecal ligation and puncture surgery in BALB/c mice. A sham-operated group was used as control (CTRL). After surgery (6 hours), CTRL and ARDS animals were assigned to receive: (1) sterile saline solution; (2) LASSBio596; (3) exogenous surfactant or (4) LASSBio596 plus exogenous surfactant (n = 22/group). RESULTS: Regardless of exogenous surfactant administration, LASSBio596 improved survival rate and reduced collagen fiber content, total number of cells and neutrophils in PLF and blood, cell apoptosis, protein content in BALF, and urea and creatinine levels. LASSBio596 plus surfactant yielded all of the aforementioned beneficial effects, as well as increased BALF lipid content and reduced surface tension. CONCLUSION: LASSBio596 exhibited major anti-inflammatory and anti-fibrogenic effects in experimental sepsis-induced ARDS. Its association with surfactant may provide further advantages, potentially by reducing surface tension.

Rats undernourished in utero have altered Ca2+ signaling and reduced fertility in adulthood.

Epidemiological and animal studies have shown that placental undernutrition impairs reproduction in adult offspring, but the underlying molecular mechanisms within the male genital tract remain unknown. Due to its special physiological characteristics in transport and the modulation of the environment to which its luminal content is exposed, we hypothesized that the vas deferens would be a highly sensitive target. The goals were to investigate whether intrauterine malnutrition affects molecular mechanisms related to Ca(2+)- and oxidative stress-modulated processes and causes structural alterations in the adult rat vas deferens that could attenuate fecundity and fertility. Male adult rats malnourished in utero had increased vas deferens weight associated with thickening of the muscular coat, a decrease in the total and haploid germ cells, a marked increase in the immature cells, and a decline in the numbers of pregnant females and total offspring per male rat. The ex vivo response of vas deferens from malnourished rats demonstrated an accentuated decrease in the contractile response to phenylephrine. The vas deferens had a marked decrease in Ca(2+) transport due to the uncoupling of Ca(2+)-stimulated ATP hydrolysis and ATP-driven Ca(2+) flux, and the downregulation of both sarco-endoplasmic reticulum Ca(2+)-ATPase 2 and the coupling factor 12-kDa FK506-binding protein. An increase in protein carbonylation (a marker of oxidative damage) and an imbalance between protein kinases C and A were observed as a legacy of undernutrition in early life. These results provide the structural and molecular basis to explain at least in part how maternal undernutrition affects fecundity and fertility in adult male rats.

Biochemical and phylogenetic analyses of phosphatidylinositol production in Angomonas deanei, an endosymbiont-harboring trypanosomatid.

BACKGROUND: The endosymbiosis in trypanosomatids is characterized by co-evolution between one bacterium and its host protozoan in a mutualistic relationship, thus constituting an excellent model to study organelle origin in the eukaryotic cell. In this association, an intense metabolic exchange is observed between both partners: the host provides energetic molecules and a stable environment to a reduced wall symbiont, while the bacterium is able to interfere in host metabolism by enhancing phospholipid production and completing essential biosynthesis pathways, such as amino acids and hemin production. The bacterium envelope presents a reduced cell wall which is mainly composed of cardiolipin and phosphatidylcholine, being the latter only common in intracellular prokaryotes. Phosphatidylinositol (PI) is also present in the symbiont and host cell membranes. This phospholipid is usually related to cellular signaling and to anchor surface molecules, which represents important events for cellular interactions. METHODS: In order to investigate the production of PI and its derivatives in symbiont bearing trypanosomatids, aposymbiotic and wild type strains of Angomonas deanei, as well as isolated symbionts, were incubated with [(3)H]myo-inositol and the incorporation of this tracer was analyzed into inositol-containing molecules, mainly phosphoinositides and lipoproteins. Gene searches and their phylogenies were also performed in order to investigate the PI synthesis in symbiontbearing trypanosomatids. RESULTS: Our results showed that the bacterium did not incorporate the tracer and that both strains produced similar quantities of PI and its derivatives, indicating that the symbiont does not influence the production of these metabolites. Gene searches related to PI synthesis revealed that the trypanosomatid genome contains an inositol transporter, PI synthase and the myo-inositol synthase. Thus, the host is able to produce PI either from exogenous myo-inositol (inositol transporter) or from myo-inositol synthesized de novo. Phylogenetic analysis using other organisms as references indicated that, in trypanosomatids, the genes involved in PI synthesis have a monophyletic origin. In accordance with experimental data, sequences for myo-inositol transport or for myo-inositol and PI biosynthesis were not found in the symbiont. CONCLUSIONS: Altogether, our results indicate that the bacterium depends on the host to obtain PI.

Early double-negative thymocyte export in Trypanosoma cruzi infection is restricted by sphingosine receptors and associated with human chagas disease.

The protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironmental and lymphoid compartments. Acute infection results in severe atrophy of the organ and early release of immature thymocytes into the periphery. To date, the pathophysiological effects of thymic changes promoted by parasite-inducing premature release of thymocytes to the periphery has remained elusive. Herein, we show that sphingosine-1-phosphate (S1P), a potent mediator of T cell chemotaxis, plays a role in the exit of immature double-negative thymocytes in experimental Chagas disease. In thymuses from T. cruzi-infected mice we detected reduced transcription of the S1P kinase 1 and 2 genes related to S1P biosynthesis, together with increased transcription of the SGPL1 sphingosine-1-lyase gene, whose product inactivates S1P. These changes were associated with reduced intrathymic levels of S1P kinase activity. Interestingly, double-negative thymocytes from infected animals expressed high levels of the S1P receptor during infection, and migrated to lower levels of S1P. Moreover, during T. cruzi infection, this thymocyte subset expresses high levels of IL-17 and TNF-α cytokines upon polyclonal stimulation. In vivo treatment with the S1P receptor antagonist FTY720 resulted in recovery the numbers of double-negative thymocytes in infected thymuses to physiological levels. Finally, we showed increased numbers of double-negative T cells in the peripheral blood in severe cardiac forms of human Chagas disease.

Mechanisms involving Ang II and MAPK/ERK1/2 signaling pathways underlie cardiac and renal alterations during chronic undernutrition.

BACKGROUND: Several studies have correlated protein restriction associated with other nutritional deficiencies with the development of cardiovascular and renal diseases. The driving hypothesis for this study was that Ang II signaling pathways in the heart and kidney are affected by chronic protein, mineral and vitamin restriction. METHODOLOGY/PRINCIPAL FINDINGS: Wistar rats aged 90 days were fed from weaning with either a control or a deficient diet that mimics those used in impoverished regions worldwide. Such restriction simultaneously increased ouabain-insensitive Na+-ATPase and decreased (Na++K+)ATPase activity in the same proportion in cardiomyocytes and proximal tubule cells. Type 1 angiotensin II receptor (AT1R) was downregulated by that restriction in both organs, whereas AT2R decreased only in the kidney. The PKC/PKA ratio increased in both tissues and returned to normal values in rats receiving Losartan daily from weaning. Inhibition of the MAPK pathway restored Na+-ATPase activity in both organs. The undernourished rats presented expanded plasma volume, increased heart rate, cardiac hypertrophy, and elevated systolic pressure, which also returned to control levels with Losartan. Such restriction led to electrical cardiac remodeling represented by prolonged ventricular repolarization parameters, induced triggered activity, early after-depolarization and delayed after-depolarization, which were also prevented by Losartan. CONCLUSION/SIGNIFICANCE: The mechanisms responsible for these alterations are underpinned by an imbalance in the PKC- and PKA-mediated pathways, with participation of angiotensin receptors and by activation of the MAPK/ERK1/2 pathway. These cellular and molecular alterations culminate in cardiac electric remodeling and in the onset of hypertension in adulthood.