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1.
Blood Transfus ; 15(1): 66-73, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27136427

RESUMO

BACKGROUND: Non-invasive foetal RHD genotyping can predict haemolytic disease of the foetus and the newborn in pregnancies with anti-D alloantibodies and also avoid antenatal anti-D prophylaxis in pregnant women carrying an RHD negative foetus. Considering that the Argentine genetic background is the result of generations of intermixing between several ethnic groups, we evaluated the diagnostic performance of a non-invasive foetal RHD determination strategy to guide targeted antenatal RhD immunoprophylaxis. This algorithm is based on the analysis of four regions of the RHD gene in cell-free foetal DNA in maternal plasma and maternal and paternal RHD genotyping. MATERIALS AND METHODS: DNA from 298 serologically D negative pregnant women between 19-28 weeks gestation were RHD genotyped. Foetal RHD status was determined by real-time PCR in 296 maternal plasma samples. In particular cases, RHDΨ and RHD-CE-Ds alleles were investigated in paternal DNA. Umbilical cord blood was collected at birth, and serological and molecular studies were performed. RESULTS: Of the 298 maternal samples, 288 were D-/RHD- and 10 D-/RHD+ (2 RHD*DAR; 5 RHD-CE-Ds; 3 RHDΨ). Plasma from RHD*DAR carriers was not analysed. Real-time PCR showed 210 RHD+ and 78 RHD- foetuses and 8 inconclusive results. In this latter group, paternal molecular studies were useful to report a RHD negative status in 5 foetuses while only 3 remained inconclusive. All the results, except one false positive due to a silent allele (RHD[581insG]), agreed with the neonatal typing performed in cord blood. DISCUSSION: The protocol used for non-invasive prenatal RHD genotyping proved to be suitable to determine foetal RHD status in our admixed population. The knowledge of the genetic background of the population under study and maternal and paternal molecular analysis can reduce the number of inconclusive results when investigating foetal RHD status.


Assuntos
Técnicas de Genotipagem/métodos , Sistema do Grupo Sanguíneo Rh-Hr/genética , DNA/sangue , DNA/genética , Feminino , Sangue Fetal/imunologia , Feto/imunologia , Feto/metabolismo , Variação Genética , Genótipo , Idade Gestacional , Humanos , Imunoterapia , Masculino , Gravidez , Diagnóstico Pré-Natal , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema do Grupo Sanguíneo Rh-Hr/imunologia
2.
Transfusion ; 54(10): 2456-62, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24819281

RESUMO

BACKGROUND: The D- phenotype is mainly caused by the complete deletion of the RHD gene in Caucasians. However, a plethora of allelic variants have been described among D- individuals from different ethnic groups. STUDY DESIGN AND METHODS: A cohort of 1314 routine serologically D- samples from white Argentineans was studied by molecular methods. RESULTS: Among the 1314 D- samples, 2.1% showed RHD-specific amplifications. One hybrid Rhesus box was detected in all D-/RHD+ samples, suggesting a hemizygous status. The RHDΨ was found in 0.7% of rr samples while DEL and null variants were detected in 16.7% of the D- samples expressing C and/or E antigens. The variants associated with the C antigen were seven RHD-CE-D(s) , two RHD(1-2)-CE(2-9)-D(10), two previously unreported RHD(329T>C)-CE(3-9)-D null alleles, one RHD(M295I), and one new RHCE(1-2)-RHD(3361del11 -10) null allele whereas those associated with the E antigen were five RHD(46T>C) and one novel RHD(581insG) null allele responsible for a premature stop codon. CONCLUSIONS: The prevalence of D-/RHD+ samples is higher than that observed in Europeans. More than 50% of the RHD alleles found were represented by RHDψ and RHD-CE-D(s) showing the African contribution to the genetic pool of the admixed population analyzed. Interestingly, three new alleles were found, two of them being hybrid structures between previously described RHD variants recombined with RHCE sequences. The knowledge of the RHD allele repertoire in our population allowed the implementation of reliable typing and transfusion strategies for a better management of patients and pregnant women.


Assuntos
Doadores de Sangue , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Argentina/epidemiologia , População Negra/genética , Doadores de Sangue/estatística & dados numéricos , Estudos de Coortes , Feminino , Frequência do Gene , Humanos , Masculino , Linhagem , Fenótipo , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Análise de Sequência de DNA , Deleção de Sequência , Testes Sorológicos , População Branca/genética
3.
Cell Logist ; 3(1): e26331, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24475373

RESUMO

AKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participate in the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1/S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.

4.
Apoptosis ; 17(5): 475-91, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22270152

RESUMO

Glucose deprivation entails oxidative stress and apoptosis in diverse cell types. Liver tissue shows high tolerance to nutritional stress, however regulation of survival in normal hepatocytes subjected to glucose restriction is unclear. We assessed the survival response of cultured hepatocytes subjected to glucose deprivation and analyzed the putative participation of protein kinase A (PKA) in this response. Six hours glucose deprivation induced a PKA dependent activation of apoptosis in cultured hepatocytes, without having an impact on non apoptotic death. Apoptotic activation associated to glucose restriction was secondary to an imbalance in cellular reactive oxygen species (ROS). In this condition, PKA inhibition led to an early prevention in mitochondrial ROS production and a late increase in scavenging enzymes transcript levels. These results supported the hypothesis that PKA could modulate glucose deprivation induced apoptotic activation by conditioning mitochondrial ROS production during glucose fasting. We presented additional evidence sustaining this model: First, glucose withdrawal led to a 95% increase in mitochondrial cAMP levels in cultured hepatocytes; second, activation of PKA significantly augmented hepatic mitochondrial ROS generation, whereas PKA inhibition elicited the opposite effect. Mitochondrial PKA signaling, previously proposed as an autonomic pathway adjusting respiration rate, emerges as a mechanism of controlling cell survival during glucose restriction.


Assuntos
Apoptose , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucose/deficiência , Hepatócitos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Caspase 3/metabolismo , Catalase/genética , Catalase/metabolismo , Sobrevivência Celular , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Citocromos c/metabolismo , Citosol/metabolismo , Hepatócitos/enzimologia , Isoquinolinas/farmacologia , L-Lactato Desidrogenase/metabolismo , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo , Transporte Proteico , Ratos , Ratos Wistar , Transdução de Sinais , Sulfonamidas/farmacologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Proteína X Associada a bcl-2/metabolismo
5.
J Cell Physiol ; 227(1): 160-71, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21374596

RESUMO

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canalículos Biliares/metabolismo , Canalículos Biliares/ultraestrutura , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imunofluorescência , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células Hep G2 , Humanos , Immunoblotting , Microscopia Confocal , Microscopia de Fluorescência
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