Caveolae-mediated endocytosis of extracellular QSOX1b modulates the migration of fibroblasts.

Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.

Participación de rafts en enfermedades neurológicas / Role of rafts in neurological disorders

Introducción: Los rafts constituyen nano-dominios estructurales de naturaleza lipoproteica que propician la eficiente transducción de señales y la modulación de procesos fisiológicos asociados a la membrana plasmática. En el sistema nervioso, la alteración de estos dominios se ha asociado con el desarrollo de diversos padecimientos. Desarrollo: En el presente artículo se revisa el concepto de rafts, los procesos del sistema nervioso en los cuales están involucrados y su papel en distintas afectaciones, entre las que se destacan las enfermedades de Parkinson, Alzheimer y Huntington. Conclusiones: Dadas las evidencias de su participación en diversas neuropatologías, la preservación y/o reconstitución de los rafts se vislumbran como una atractiva estrategia terapéutica.(AU)

Introduction: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. Development: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. Conclusions: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.(AU)

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Role of rafts in neurological disorders.

INTRODUCTION: Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders. DEVELOPMENT: We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease. CONCLUSIONS: Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.

Local Ca2+ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells.

An increase in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-sensitive enzymes such as Ca2+/calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca2+ channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca2+]i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca2+ signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca2+ chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca2+ events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca2+ signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.

Increased TMEM16A-Mediated Ca2+-Activated Cl- Currents in Portal Vein Smooth Muscle Cells of Caveolin 1-Deficient Mice.

Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.

Deformation of caveolae impacts global transcription and translation processes through relocalization of cavin-1.

Caveolae are invaginated membrane domains that provide mechanical strength to cells in addition to being focal points for the localization of signaling molecules. Caveolae are formed through the aggregation of caveolin-1 or -3 (Cav1/3), membrane proteins that assemble into multifunctional complexes with the help of caveola-associated protein cavin-1. In addition to its role in the formation of caveolae, cavin-1, also called polymerase I and transcript release factor, is further known to promote ribosomal RNA transcription in the nucleus. However, the mechanistic link between these functions is not clear. Here, we found that deforming caveolae by subjecting cells to mild osmotic stress (150-300 mOsm) changes levels of GAPDH, Hsp90, and Ras only when Cav1/cavin-1 levels are reduced, suggesting a link between caveola deformation and global protein expression. We show that this link may be due to relocalization of cavin-1 to the nucleus upon caveola deformation. Cavin-1 relocalization is also seen when Cav1-Gαq contacts change upon stimulation. Furthermore, Cav1 and cavin-1 levels have been shown to have profound effects on cytosolic RNA levels, which in turn impact the ability of cells to form stress granules and RNA-processing bodies (p-bodies) which sequester and degrade mRNAs, respectively. Our studies here using a cavin-1-knockout cell line indicate adaptive changes in cytosolic RNA levels but a reduced ability to form stress granules. Taken together, our findings suggest that caveolae, through release of cavin-1, communicate extracellular cues to the cell interior to impact transcriptional and translational.

Caveolae-Associated Molecules, Tumor Stroma, and Cancer Drug Resistance: Current Findings and Future Perspectives.

The discovery of small, "cave-like" invaginations at the plasma membrane, called caveola, has opened up a new and exciting research area in health and diseases revolving around this cellular ultrastructure. Caveolae are rich in cholesterol and orchestrate cellular signaling events. Within caveola, the caveola-associated proteins, caveolins and cavins, are critical components for the formation of these lipid rafts, their dynamics, and cellular pathophysiology. Their alterations underlie human diseases such as lipodystrophy, muscular dystrophy, cardiovascular disease, and diabetes. The expression of caveolins and cavins is modulated in tumors and in tumor stroma, and their alterations are connected with cancer progression and treatment resistance. To date, although substantial breakthroughs in cancer drug development have been made, drug resistance remains a problem leading to treatment failures and challenging translation and bench-to-bedside research. Here, we summarize the current progress in understanding cancer drug resistance in the context of caveola-associated molecules and tumor stroma and discuss how we can potentially design therapeutic avenues to target these molecules in order to overcome treatment resistance.

Monitoring Transmembrane and Peripheral Membrane Protein Interactions by Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy.

Caveolae are bulb-shaped invaginations of the plasma membrane that are enriched in specific lipids including cholesterol, phosphatidylserine and sphingolipids. Caveolae have many described cellular roles and functions, including endocytic transport, transcytosis, mechanosensing, and serving as a buffer against plasmalemmal stress. Caveola are formed through interactions between integral membrane proteins (Caveolin) and a cavin family of peripheral proteins (Cavins). Nearly half of the human proteome resides within or at the surface of membranes. Studying protein-protein interactions, especially of transmembrane domain containing proteins can be challenging. Fortunately, sophisticated biophysical methods allow for the monitoring of protein interactions in intact cells. Here, we describe the principles of Förster resonance energy transfer, fluorescence lifetime, and how their properties can be used to assess protein-protein interactions. Additionally, we discuss and demonstrate how fluorescence lifetime can be monitored microscopically thereby providing caveolin-cavin interaction data from living cells.

The interactions between dietary fats intake and Caveolin 1 rs 3807992 polymorphism with fat distribution in overweight and obese women: a cross-sectional study.

BACKGROUND: It has been reported that dietary fats and genetic factors in individuals are associated with the pattern of fat distribution. This study aimed to evaluate the interaction between dietary fats intake and Caveolin1 (CAV-1) rs 3807s992 polymorphism with fat distribution in overweight and obese women. METHODS: A total of 221 participants were included in the current cross-sectional study. Body composition, biochemical parameters were evaluated by body composition analyzer and Pars Azmoon kits and genotypes determination was performed by PCR-RFLP, dietary fats were measured using a validated semi-quantitative food frequency questionnaire (FAQ). RESULTS: The frequency of GG, AA and AG genotypes were 53.1, 24.6, and 22.3%, respectively, and the mean intake of total dietary fat intake was 97.47 ± 36.87 g. There was positive significant interaction between total fat intake and AA genotype on visceral fat level (p = 0.001), trunk fat (p = 0.01) and waist circumference (p = 0.05), positive significant interaction between total fat intake and AG genotype on the waist to hip ratio (WHR) (p = 0.02) and visceral fat level (p = 0.05), positive borderline significant interaction between saturated fatty acid and AA genotype on the trunk fat (p = 0.06), and between trans-fatty acids and AG genotype on WHR (p = 0.04), visceral fat level (p = 0.01), and between monounsaturated fatty acid and AG genotype on WHR (p = 0.04), and a borderline interaction between polyunsaturated fatty acid and AA genotypes on visceral fat level (p = 0.06), negative significant interaction between AG genotypes and linolenic acid on WHR (p = 0.04), borderline significant interaction between ALA and AG genotype on WHR (p = 0.06). CONCLUSIONS: Our findings showed that CAV-1 rs 3807992 polymorphism and dietary fats were associated with fat distributions in individuals.

Role of rafts in neurological disorders. / Participación de rafts en enfermedades neurológicas.

INTRODUCTION: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. DEVELOPMENT: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. CONCLUSIONS: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.

Membrane Curvature, Trans-Membrane Area Asymmetry, Budding, Fission and Organelle Geometry.

In biology, the modern scientific fashion is to mostly study proteins. Much less attention is paid to lipids. However, lipids themselves are extremely important for the formation and functioning of cellular membrane organelles. Here, the role of the geometry of the lipid bilayer in regulation of organelle shape is analyzed. It is proposed that during rapid shape transition, the number of lipid heads and their size (i.e., due to the change in lipid head charge) inside lipid leaflets modulates the geometrical properties of organelles, in particular their membrane curvature. Insertion of proteins into a lipid bilayer and the shape of protein trans-membrane domains also affect the trans-membrane asymmetry between surface areas of luminal and cytosol leaflets of the membrane. In the cases where lipid molecules with a specific shape are not predominant, the shape of lipids (cylindrical, conical, or wedge-like) is less important for the regulation of membrane curvature, due to the flexibility of their acyl chains and their high ability to diffuse.

Encephalitic Alphaviruses Exploit Caveola-Mediated Transcytosis at the Blood-Brain Barrier for Central Nervous System Entry.

Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

CaMKII Modulates the Cardiac Transient Outward K+ Current through its Association with Kv4 Channels in Non-Caveolar Membrane Rafts.

BACKGROUND/AIMS: To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. METHODS: Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. RESULTS: Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CONCLUSION: CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.

Protective Effect of Tocotrienol on In Vitro and In Vivo Models of Parkinson's Disease.

Parkinson's disease (PD) is a common progressive neurodegenerative disease. It has been reported that oxidative stress contributes, at least in part, to its pathogenesis. Although dietary epidemiological studies suggest that sufficient intake of vitamin E may prevent the onset of PD, antioxidative therapy for PD with exogenous antioxidants involving α-tocopherol has not been successful in the clinical setting thus far. In recent years, the non-antioxidant activities of vitamin E have been given attention to. In the present study, to determine the antioxidant-independent cytoprotective activity of vitamin E, we investigated whether tocotrienols (T3s), another members of vitamin E family, exhibit the neuroprotective effect in cell and mouse models of PD independently of their antioxidant activities. Treatment with T3s, especially γ- and δ-T3s, exhibited cytoprotective effects via activation of PI3K/Akt signaling pathway in a cellular PD model. We also identified estrogen receptor (ER) ß as an upstream mediator of PI3K/Akt signaling and demonstrated the direct binding of T3 to ERß in vitro. Silencing expression of caveolin suppressed the cytoprotective effects of T3, indicating that caveola formation plays an important role in the cytoprotection by T3 via ERß/PI3K/Akt signaling pathway. Thus it has been shown that T3 exerts cytoprotective function by a novel mechanism, which includes membrane ERß/PI3K/Akt signaling via caveola formation as well as its antioxidant activity. Furthermore, we revealed that δ-T3 treatment relieved PD-related symptoms in PD model mice. These results suggest that T3 elicits the cytoprotective effects via ERß/PI3K/Akt signaling pathway in cellular and murine PD models.

[The role of caveolae in cataractogenesis: examination of human lens epithelial cells]. / Caveolák szerepe a szürke hályog képzodésében: humán szemlencse epithelsejtjeinek vizsgálata.

Inrtoduction: Caveolae are flask shaped with 50-100 nm size, non-clathrin associated invaginations of the plasmamembrane. The main membrane protein of the structures is caveolin-1. Caveolae play an important role in numerous cellular functions including vesicular transport and cell-cycle regulation, and create platforms for classical and alternative signaling pathways. According to international studies, caveolae may influence the physiology and pathology of lens epithelial cells. AIM: The aim of the study was to examine and compare the morphology of caveolae and the immunohistochemical difference of caveolin-1 in control (myopic and hyperopic) lens epithelial cells and human lens epithelial cells affected by cataract. Authors investigated whether caveolae might have a role in cataractogenesis. METHOD: Anterior lens capsules were obtained by capsulorhexis during surgery of senile cataract and refractive surgery of the clear lens. Ultra-fine sections have been studied by transmission electron microscopy, and semi-fine samples were labelled for immunohistochemistry with polyclonal caveolin-1 and cavin-1 antibodies. RESULTS: By immunohistochemistry, in the control group, significant caveolin-1 label with low cavin-1 signal were measured in the lens epithelial cells. In the cataract group high cavin-1 and caveolin-1 expression was detected. In the control group, caveolae were not observed, but in the lens epithelial cells with cataract, increased number of caveolae have been detected by electron microscopy. CONCLUSIONS: For the development and maintenance of the specific caveolae shape, caveolin-1 is needed to be accompanied by cavin-1. Therefore, it is presumable that the increased expression of cavin-1 could explain the higher number of caveolae in the cataract group. These results might suggest that caveolae might play a role in cataractogenesis. Orv Hetil. 2019; 160(8): 300-308.

Long QT syndrome caveolin-3 mutations differentially modulate Kv 4 and Cav 1.2 channels to contribute to action potential prolongation.

KEY POINTS: Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 channels responsible for Ito . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating ICa,L but, for Cav3-S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches. ABSTRACT: Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Cav 1.2+Cav ß2cN4 channels, as well as Kv 4.2 and Kv 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca2+ -dependent inactivation of ICa,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+ -dependent inactivation of ICa,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future.

Characterization of Caveola-Vesicle Complexes (CVCs) Protein, PHIST/CVC-8195 in Plasmodium vivax.

Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, PcyPHIST/CVC-8195, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about PHIST/CVC-8195 protein in P. vivax. In this study, the recombinant PvPHIST/CVC-8195 N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of PvPHIST/CVC-8195 N and C termini in blood stage parasites was also determined. The antigenicity of recombinant PvPHIST/CVC-8195 N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of PvPHIST/CVC-8195 which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of PvPHIST/CVC-8195. These results suggest that the PvPHIST/CVC-8195 is localized on the CVCs and may be immunogenic in natural infection of P. vivax.

Characterization of Caveola-Vesicle Complexes (CVCs) Protein, PHIST/CVC-81₉₅ in Plasmodium vivax

Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, PcyPHIST/CVC-81₉₅, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about PHIST/CVC-81₉₅ protein in P. vivax. In this study, the recombinant PvPHIST/CVC-81₉₅ N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of PvPHIST/CVC-81₉₅ N and C termini in blood stage parasites was also determined. The antigenicity of recombinant PvPHIST/CVC-81₉₅ N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of PvPHIST/CVC-81₉₅ which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of PvPHIST/CVC-81₉₅. These results suggest that the PvPHIST/CVC-81₉₅ is localized on the CVCs and may be immunogenic in natural infection of P. vivax.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...