RESUMO
SVEP1, also known as Polydom, is a large extracellular mosaic protein with functions in protein interactions and adhesion. Since Svep1 knockout animals show severe edema and lymphatic system malformations, the aim of this study is to evaluate the presence of SVEP1 variants in patients with lymphedema. We analyzed DNA from 246 lymphedema patients for variants in known lymphedema genes, 235 of whom tested negative and underwent a second testing for new candidate genes, including SVEP1, as reported here. We found three samples with rare heterozygous missense single-nucleotide variants in the SVEP1 gene. In one family, healthy members were found to carry the same variants and reported some subclinical edema. Based on our findings and a review of the literature, we propose SVEP1 as a candidate gene that should be sequenced in patients with lymphatic malformations, with or without lymphedema, in order to investigate and add evidence on its possible involvement in the development of lymphedema.
Assuntos
Anormalidades Linfáticas , Linfedema , Moléculas de Adesão Celular , Humanos , Linfangiogênese/genética , Anormalidades Linfáticas/diagnóstico , Anormalidades Linfáticas/genética , Sistema Linfático/metabolismo , Linfedema/diagnóstico , Linfedema/genética , Linfedema/metabolismo , MorfogêneseRESUMO
SEMA3A is a semaphorin involved in cell signaling with PlexinA1 and Neuropilin-1 (NRP1) receptors and it is responsible for recruiting dendritic cells into lymphatics. Mutations in the SEMA3A gene result in abnormalities in lymphatic vessel development and maturation. We investigated the association of SEMA3A variants detected in lymphedema patients with lymphatic maturation and lymphatic system malfunction. First, we used NGS technology to sequence the SEMA3A gene in 235 lymphedema patients who carry wild type alleles for known lymphedema genes. We detected three different missense variants in three families. Bioinformatic results showed that some protein interactions could be altered by these variants. Other unaffected family members of the probands also reported different episodes of subclinical edema. We then evaluated the importance of the SEMA3A gene in the formation and maturation of lymphatic vessels. Our results determined that SEMA3A variants segregate in families with lymphatic system malformations and recommend the inclusion of SEMA3A in the gene panel for testing of patients with lymphedema.
Assuntos
Linfangiogênese/genética , Vasos Linfáticos/metabolismo , Linfedema/etiologia , Linfedema/metabolismo , Semaforina-3A/genética , Animais , Biologia Computacional/métodos , Suscetibilidade a Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Humanos , Linfedema/diagnóstico , Semaforina-3A/metabolismoRESUMO
CYP26B1 is a member of the cytochrome P450 family and is responsible for the break-down of retinoic acid for which appropriate levels are important for normal development of the cardiovascular and lymphatic systems. In a cohort of 235 patients with lymphatic malformations, we performed genetic testing for the CYP26B1 gene. These probands had previously tested negative for known lymphedema genes. We identified two heterozygous missense CY-P26B1 variants in two patients. Our bioinformatic study suggested that alterations caused by these variants have no major effect on the overall stability of CYP26B1 protein structure. Balanced levels of retinoic acid maintained by CYP26B1 are crucial for the lymphatic system. We identified that CYP26B1 could be involved in predisposition for lymphedema. We propose that CYP26B1 be further explored as a new candidate gene for genetic testing of lymphedema patients.
Assuntos
Linfangiogênese , Linfedema/patologia , Mutação de Sentido Incorreto , Ácido Retinoico 4 Hidroxilase/genética , Feminino , Humanos , Linfedema/genética , Linfedema/metabolismo , Pessoa de Meia-Idade , Prognóstico , Conformação Proteica , Ácido Retinoico 4 Hidroxilase/química , Ácido Retinoico 4 Hidroxilase/metabolismoRESUMO
Primary lymphedema is characterized by altered morphological development of lymphatic vessels causing fluid accumulation in interstitial spaces. In familial forms, it is primarily transmitted as a dominant Mendelian trait with heterozygous mutations in genes involved in lymphangiogenesis. We used PCR and direct sequencing to analyze the region of the fms-related tyrosine kinase 4 (FLT4) gene encoding the "tyrosine-kinase domain" and the single exon of the forkhead box C2 (FOXC2) gene in 46 Italian probands with primary lymphedema, 42 of whom had familial forms. We identified 12 mutations in 12 patients (12/46, 26%), six in the FLT4 gene and six in the FOXC2 gene. Most of the mutations (9/12, 75%) were new, and none were identified in 100 healthy subjects or listed in the NCBI dbSNP. A clear relation emerged between genotype and phenotype because 4/5 (80%) probands with onset at birth showed FLT4 mutations and 4/5 (80%) probands without distichiasis and with FOXC2 mutations had an amino-acid substitution outside the forkhead domain. Besides the allelic heterogeneity shown by unique mutations in each proband, the absence of mutations in almost 75% of familial cases of primary lymphedema also suggests genetic heterogeneity.
Assuntos
Fatores de Transcrição Forkhead/genética , Linfangiogênese/genética , Linfedema/genética , Mutação , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Idade de Início , Estudos de Casos e Controles , Análise Mutacional de DNA , Éxons , Feminino , Predisposição Genética para Doença , Humanos , Itália , Linfedema/patologia , Linfedema/fisiopatologia , Masculino , Fenótipo , Reação em Cadeia da Polimerase , Adulto JovemRESUMO
Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.
Assuntos
Humanos , Recém-Nascido , Eritrócitos , Hemólise/efeitos dos fármacos , Chumbo , Metais , Alumínio , Cádmio , Volume de Eritrócitos , Eritrócitos , Sangue Fetal , Íons , Concentração Osmolar , ZincoRESUMO
Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.
Assuntos
Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Chumbo/farmacologia , Metais/farmacologia , Alumínio/farmacologia , Cádmio/farmacologia , Volume de Eritrócitos , Eritrócitos/fisiologia , Sangue Fetal , Humanos , Recém-Nascido , Íons/farmacologia , Concentração Osmolar , Zinco/farmacologiaRESUMO
In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.
Assuntos
Humanos , Feminino , Gravidez , Membrana Celular , Eritrócitos , Lítio , ATPase Trocadora de Sódio-Potássio , Transporte de Íons/fisiologia , Amilorida , Membrana Celular , Meios de Cultura , Eritrócitos , Etilmaleimida , Sangue Fetal , Hematopoese , Cinética , Sódio , ATPase Trocadora de Sódio-Potássio , Transporte de Íons/efeitos dos fármacosRESUMO
Neonatal red cells (umbilical cord blood) were in vitro incubated in isotonic media (thiocyanate as predominant anion). This experimental condition was selected as it was known that this chaotropic anion induced activation of Na+-H+ exchange. The transport amiloride-sensitive mechanism, that decreased the acidic intracellular pH change occurring in this medium, would correspond to Na+-H+ exchange (NHE1 isoform). However, the Na+-Li+ exchange, also determined in the cells in the above-mentioned medium was not affected by amiloride. The present data suggest that an amiloride insensitive Na+-H+ exchange isoform would express Na+-Li+ countertransport in these cells.
Assuntos
Eritrócitos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Amilorida/farmacologia , Cátions Monovalentes , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Humanos , Recém-Nascido , Transporte de Íons , Lítio/metabolismo , Isoformas de Proteínas/metabolismo , Fatores de TempoRESUMO
The plasma membrane Na+/Li+ exchange is of research interest because it is involved in some pathologic states. A kinetic analysis of this transport system would be of greater importance as compared to the flux determination under "standard" conditions (reactants saturating levels) as a way to define normal values. Unidirectional lithium fluxes in red blood cells from normal adults were determined as a function of "trans" (contralateral) sodium ion concentration. Relating Li+ efflux and influx (Na+ "trans") kinetic parameters we found free carrier reorientation between the two phases (asymmetry).
Assuntos
Eritrócitos/metabolismo , Lítio/metabolismo , Sódio/metabolismo , Cátions Monovalentes , Células Cultivadas , Humanos , Transporte de Íons , Cinética , Potássio/metabolismo , Espectrofotometria AtômicaRESUMO
Li/Na exchange kinetic parameters (Km, Vmax) were determined in red blood cells from normal adolescents with or without hypertensive antecessors, as well as plasmatic levels of high density lipoproteins. In red blood cells from adolescents with hypertensive antecessors, we observed significantly lower Km values compared to the other group. HDL-c (high density lipoprotein-cholesterol) plasmatic values did not differ significantly. Furthermore, in normal adolescents without hypertensive ancestors, the Vmax significantly correlated with the HDL-c plasmatic values. The results indicated that the Km parameter of this transport mechanism is affected by genetic factors and data are also presented to demonstrate that the Vmax parameter is affected by environmental (cellular) factors. It is suggested that Li/Na exchange Km values would be a risk index of essential hypertension development.
Assuntos
Eritrócitos/metabolismo , Hipertensão/genética , Lítio/metabolismo , Sódio/metabolismo , Adolescente , Cátions Monovalentes , HDL-Colesterol/sangue , Humanos , Hipertensão/sangue , Transporte de Íons , Cinética , Modelos LinearesRESUMO
In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.
Assuntos
Membrana Celular/metabolismo , Eritrócitos/metabolismo , Transporte de Íons/fisiologia , Lítio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Amilorida/farmacologia , Membrana Celular/efeitos dos fármacos , Meios de Cultura , Eritrócitos/efeitos dos fármacos , Etilmaleimida/farmacologia , Feminino , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/metabolismo , Hematopoese/fisiologia , Humanos , Transporte de Íons/efeitos dos fármacos , Cinética , Gravidez , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
Human red blood cells from prenatal stages of hemopoiesis (umbilical cord blood) and with increased lithium content were submitted to media with varying sodium concentrations in order to characterize extracellular sodium dependent-lithium efflux. On the other hand cells with different sodium contents were submitted to lithium media in order to characterize cellular sodium dependent-lithium influx through the above system. Experimental data were fitted by simple Michaelis-Menten kinetics. The Km value for lithium efflux (extracellular sodium-dependent) was significantly lower than the Km value for lithium influx (cellular sodium -dependent). The Vmax value for lithium influx was significantly greater than that for lithium efflux. The Vmax/Km ratio values were independent from the way in which the fluxes were measured. It is concluded that the simple carrier cannot be excluded as a model of the Na+-Li+ exchange system in these cells.
Assuntos
Eritrócitos/citologia , Eritrócitos/enzimologia , Hematopoese/fisiologia , Lítio/farmacocinética , Sódio/farmacocinética , Adulto , Fatores Etários , Transporte Biológico/fisiologia , Eritrócitos/química , Sangue Fetal/fisiologia , Humanos , Recém-Nascido , Cinética , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The kinetic parameters of Na(+)-Li+ exchange were studied in both neonatal and adult's red blood cells in order to evaluate if the asymmetry for Li+ fluxes (Na(+)-contralateral-dependent) is expressed in both types of cells. Maximum velocities (Vmax) and Km (half-activation constant) were measured for Li+ fluxes in both types of cells. In human neonatal red blood cells (nRBC), extracellular Na+-dependent Li+ efflux was shown to be a saturable function of external sodium concentration with high affinity (apparent Km: 2.1 mM) and low capacity (maximal velocity Vmax = 0.3 mmoles Li+ 1('''10 cells h(-1)). The Vmax and apparent Km for cellular Na(+)-dependent Li+ influx were higher (Km = 12.9 mM; Vmax = 1.07 mmoles Li+ 1(-1) cells h(-1)). The results provide evidence for intrinsic functional asymmetry in this transport system, as the transporter is more prevalent and stable in the inward-facing conformation. These kinetic observations may be explained in terms of the simple carrier transport model. Similar findings were found for this system in human adult's red cells. These results point to the asymmetry pattern (related to Na+ activation of Li+ flux) as developed in red cells from late prenatal stages of hemopoiesis.
Assuntos
Eritrócitos/fisiologia , Hematopoese , Lítio/metabolismo , Sódio/metabolismo , Adulto , Células da Medula Óssea/citologia , Eritrócitos/metabolismo , Sangue Fetal , Humanos , Técnicas In Vitro , Recém-Nascido , Cinética , Fígado/citologia , Fígado/embriologia , Potássio/metabolismo , Baço/citologia , Baço/embriologiaRESUMO
An attenuation (or reversion) of the prolytic effect of lead on neonatal red cells is observed in iso- or hypotonic low ionic strength media. This effect correlates neither with concomitant activation of K+ (Ca2+ or Pb2+) channels nor with volume reduction. Neonatal erythrocytes were used in this study owing to their greater cellular density, as compared with adult red cells, for the above mentioned channels. The attenuation-reversion effect would be mediated through lead interactions with the cytoskeleton, a structure that is the limiting factor for red cell lysis in low ionic strength media.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Chumbo/toxicidade , Canais de Potássio/efeitos dos fármacos , Contagem de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Recém-Nascido , Concentração OsmolarRESUMO
Red cells from umbilical cord with increased lithium content were submitted to different experimental conditions in order to study lithium flux components. There appeared three components: First, an ouabain-sensitive component, related to Na+ replacement with Li+ in the primary active Na+/K+ transport system. The magnitude of this fraction is greater than in adults' red cells. Second, an outside sodium-dependent Li+ efflux fraction, corresponding to the Li+/Na+ countertransport system with Vmax and K(m) values of 0.1 (mmol/l cells.h) and 2.58 (mmol/l), respectively. The Na+o-affinity for lithium efflux in this system is greater in neonatal than in adults' red cells. Third, a leak fraction with an equal value to that reported in adults' red cells. Furthermore, the possible non-existence of a bumetanide-sensitive lithium flux fraction was shown in neonatal red cells.
Assuntos
Eritrócitos/química , Hematopoese , Lítio/sangue , Adulto , Feminino , Sangue Fetal/química , Hematócrito , Humanos , Técnicas In Vitro , Cinética , Sódio/sangueRESUMO
Differences in ionic transport mechanisms through human red cell membranes from pre and postnatal haemopoietic stages of development have been related. The analysis of calcium-sensitive conductive potassium pathways [K+ (Ca2+)] was realized from intracellular calcium level increase after treatment of cells with ionophores. The effect of calcium ionophore (A23187) on potassium transport in human neonatal red cells (nRC) was studied and compared with red cells from adults (aRC). Analysis focused on intra/extracellular potassium redistribution and potassium conductance [K+ (Ca2+)] values in incubated red cells suspensions. A difference was observed between neonatal and adult's red cells only for the first of the parameters referred to. Conductance values of the same order of magnitude for these potassium transport pathways between both cell types suggest that the results presented could rest on different [K+ (Ca2+)] cellular density.
Assuntos
Envelhecimento/sangue , Calcimicina/farmacologia , Cálcio/metabolismo , Eritrócitos/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Ionóforos/farmacologia , Trifosfato de Adenosina/sangue , Adulto , Contagem de Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Sangue Fetal/efeitos dos fármacos , Humanos , Recém-Nascido , Transporte de Íons/efeitos dos fármacos , Potássio/metabolismoRESUMO
The effect of replacement of chloride by thiocyanate has been investigated in neonatal human red cells. On incubating these cells in isotonic SCN- medium, a rapid cellular swelling was observed, which was not evident in Cl- media. Incubation in hypertonic SCN- medium showed a rapid osmotic shrinkage followed by reswelling back to the initial volume. This regulatory volume increase was completed in a shorter time than in Cl- medium. Whole water accumulated by the cells in both experimental conditions can be accounted for by net Na+ uptake (twofold augmentation with respect to initial value). Amiloride inhibits specifically both cellular swelling and the Na+ content increase in these experimental conditions. Similar experiments in neonatal red cells incubated in NO3- media, as well as in adult red cells placed in NO3- or SCN- media, did not exhibit the same response. The findings suggest that the response of neonatal red cells in a SCN- containing media was mediated by activation of a Na+/H+ antiport mechanism.
Assuntos
Cloretos/sangue , Eritrócitos/citologia , Tiocianatos/sangue , Adulto , Amilorida/farmacologia , Ânions , Eritrócitos/efeitos dos fármacos , Humanos , Soluções Hipertônicas , Soluções Hipotônicas , Recém-Nascido , Sódio/sangue , Trocadores de Sódio-Hidrogênio/sangueRESUMO
A "pulse like" increase of cytoplasmic calcium concentration, which is proportional to ionophore concentration, is induced in red cells by exposure to A23187. Different Ca2+ levels are attained depending on cellular calcium buffering power and/or primary active calcium transport activation. We examined the effect of A23187 concentration of potassium loss in neonatal (nRC) as well as in adult red cells (aRC). The increase in ionophore concentration produced an "all- or -none" recruitment in adult cells and a "gradual" one in neonatal red cells. The "gradual" response observed in nRC would suggest that the "all or none" character of the response is not present in red cells during the foetal stages of haematopoiesis.
Assuntos
Calcimicina/farmacologia , Eritrócitos/metabolismo , Sangue Fetal/metabolismo , Potássio/sangue , Trifosfato de Adenosina/sangue , Adulto , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Espaço Extracelular/metabolismo , Sangue Fetal/efeitos dos fármacos , Humanos , Potássio/metabolismo , Sódio/sangue , Fatores de TempoRESUMO
Human fetal red cells show heterogeneity of 3H-ouabain binding sites. These cells were chosen as a model to look into unconjugated bilirubin effects on the primary active Na(+)-K+ transport mechanism. Evidences are presented suggesting that unconjugated bilirubin affects 3H-ouabain binding but not through a direct effect. This is supported by the fact that the "low affinity" subgroup sites of the last mentioned ligand persists after unconjugated bilirubin treatment of cells, whereas the "high-affinity" subgroup disappears.
Assuntos
Bilirrubina/farmacologia , Eritrócitos/efeitos dos fármacos , Sangue Fetal/citologia , Ouabaína/metabolismo , ATPase Trocadora de Sódio-Potássio/sangue , Sítios de Ligação , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Humanos , Ligantes , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
To study some characteristics of calcium-activated potassium channels free from plasma membrane lipid perturbations, neonatal red blood cells were selected. To this end, cells not previously treated (ATP depletion and/or reversible lysis) were exposed to the ionophore A23187 in a NO3 medium with calcium. The net efflux of potassium from the cells was studied. Preincubation in a medium devoid of potassium induced a significant decrease only in the rate constant of potassium flux. The data suggest that, in the absence of plasma membrane lipid impairment, changes in internal sites reactivity with calcium ions of channel proteins would be involved in the refractoriness. This would be at variance with channel density modifications.