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1.
Braz J Med Biol Res ; 54(7): e10579, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34008754

RESUMEN

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cóclea , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Células Ciliadas Auditivas , Ratones , Órgano Espiral , Receptores Notch , Factor de Transcripción HES-1/genética
2.
Braz. j. med. biol. res ; 54(7): e10579, 2021. tab, graf
Artículo en Inglés | LILACS | ID: biblio-1249313

RESUMEN

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.


Asunto(s)
Animales , Ratas , Cóclea , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Órgano Espiral , Diferenciación Celular , Receptores Notch , Factor de Transcripción HES-1/genética , Células Ciliadas Auditivas
3.
Braz J Med Biol Res ; 42(2): 168-71, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19274344

RESUMEN

Mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of nonsyndromic recessive hearing loss in many countries. We report here on a novel point mutation in GJB2, p.L76P (c.227C>T), in compound heterozygosity with a c.35delG mutation, in two Brazilian sibs, one presenting mild and the other profound nonsyndromic neurosensorial hearing impairment. Their father, who carried a wild-type allele and a p.L76P mutation, had normal hearing. The mutation leads to the substitution of leucine (L) by proline (P) at residue 76, an evolutionarily conserved position in Cx26 as well as in other connexins. This mutation is predicted to affect the first extracellular domain (EC1) or the second transmembrane domain (TM2). EC1 is important for connexon-connexon interaction and for the control of channel voltage gating. The segregation of the c.227C>T (p.L76P) mutation together with c.35delG in this family indicates a recessive mode of inheritance. The association between the p.L76P mutation and hearing impairment is further supported by its absence in a normal hearing control group of 100 individuals, 50 European-Brazilians and 50 African-Brazilians.


Asunto(s)
Conexinas/genética , Sordera/genética , Genes Recesivos/genética , Mutación Missense/genética , Brasil , Niño , Preescolar , Conexina 26 , Sordera/etnología , Familia , Femenino , Humanos , Masculino
4.
Braz. j. med. biol. res ; 42(2): 168-171, Feb. 2009. ilus
Artículo en Inglés | LILACS | ID: lil-506883

RESUMEN

Mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of nonsyndromic recessive hearing loss in many countries. We report here on a novel point mutation in GJB2, p.L76P (c.227C>T), in compound heterozygosity with a c.35delG mutation, in two Brazilian sibs, one presenting mild and the other profound nonsyndromic neurosensorial hearing impairment. Their father, who carried a wild-type allele and a p.L76P mutation, had normal hearing. The mutation leads to the substitution of leucine (L) by proline (P) at residue 76, an evolutionarily conserved position in Cx26 as well as in other connexins. This mutation is predicted to affect the first extracellular domain (EC1) or the second transmembrane domain (TM2). EC1 is important for connexon-connexon interaction and for the control of channel voltage gating. The segregation of the c.227C>T (p.L76P) mutation together with c.35delG in this family indicates a recessive mode of inheritance. The association between the p.L76P mutation and hearing impairment is further supported by its absence in a normal hearing control group of 100 individuals, 50 European-Brazilians and 50 African-Brazilians.


Asunto(s)
Niño , Preescolar , Femenino , Humanos , Masculino , Conexinas/genética , Sordera/genética , Genes Recesivos/genética , Mutación Missense/genética , Brasil , Sordera/etnología , Familia
5.
Biochem Biophys Res Commun ; 343(3): 675-6, 2006 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-16574076

RESUMEN

Ballana et al. [E. Ballana, E. Morales, R. Rabionet, B. Montserrat, M. Ventayol, O. Bravo, P. Gasparini, X. Estivill, Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment, Biochem. Biophys. Res. Commun. 341 (2006) 950-957] detected a T1291C mutation segregating in a Cuban pedigree with hearing impairment. They interpreted it as probably pathogenic, based on family history, RNA conformation prediction and its absence in a control group of 95 Spanish subjects. We screened a sample of 203 deaf subjects and 300 hearing controls (110 "European-Brazilians" and 190 "African-Brazilians") for the mitochondrial mutations A1555G and T1291C. Five deaf subjects had the T1291C substitution, three isolated cases and two familial cases. In the latter, deafness was paternally inherited or segregated with the A1555G mutation. This doesn't support the hypothesis of T1291C mutation being pathogenic. Two "African-Brazilian" controls also had the T1291C substitution. Six of the seven T1291C-carriers (five deaf and two controls) had mitochondrial DNA of African origin, belonging to macrohaplogroup L1/L2. Therefore, these data point to T1291C substitution as most probably an African non-pathogenic polymorphism.


Asunto(s)
Sordera/genética , Polimorfismo de Nucleótido Simple , ARN Ribosómico/genética , Secuencia de Bases , Población Negra/genética , Brasil/etnología , ADN Mitocondrial/química , Sordera/etnología , Genes Mitocondriales , Genes de ARNr , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Penetrancia , Mutación Puntual , ARN Ribosómico/química , Alineación de Secuencia , Población Blanca/genética
6.
Immunohematology ; 17(4): 111-6, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-15373576

RESUMEN

Serologic ABO blood typing is routinely performed using anti-A and anti-B sera to distinguish four phenotypes (A, B, AB, and O). Restriction fragment length polymorphisms (RFLPs) and DNA sequence studies offer the possibility of direct ABO genotyping. We used polymerase chain reaction-RFLP analysis to determine the frequency of O(1) and O(2) alleles in 82 unrelated blood donors in São Paulo, Brazil, known to be group O. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. Different genotypes (O(1)O(1), O(1)O(2), O(2)O(2)) were identified after digestion with restriction enzymes KpnI, HpaII, and AluI, followed by agarose gel electrophoresis. Of 82 samples analyzed, 74 were O(1)O(1), 7 were O(1)O(2), and 1 was O(2)O(2). These results showed the frequency of O(1)O(1), O(1)O(2), and O(2)O(2) genotypes to be 90.24 percent, 8.53 percent, and 1.22 percent, respectively, in blood donors in São Paulo, Brazil.

7.
Rev Inst Med Trop Sao Paulo ; 41(4): 235-8, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10564917

RESUMEN

We studied 12 Hb C carriers: 4 homozygotic Hb CC and 8 heterozygotic. We observed the presence of free crystals in the peripheral blood of the homozygotes but in none of the heterozygotes. However, after incubation with 3% NaCl we were able to detect crystals in the heterozygotes (Hb AC and Hb SC), and in the homozygotes (Hb CC). In patient 04 (P04) less crystals formation occurred due to inhibition of the process by the presence of elevated levels of Hb F (12.2%). All the homozygotic patients had a splenomegaly of 3 to 6 fingerbreadths. We believe that the spleen wears off with time, thus allowing the passage of crystals to the peripheral blood. This finding might be associated with splenic insufficiency without a reduction of its dimensions. Finally, the finding of crystals in the peripheral blood permitted the diagnosis of Hb C obviating the need for electrophoresis.


Asunto(s)
Enfermedad de la Hemoglobina C/diagnóstico , Hemoglobina C/química , Esplenomegalia/sangre , Cristalización , Electroforesis en Gel de Agar , Homocigoto , Humanos , Bazo/fisiopatología
8.
Rev. Inst. Med. Trop. Säo Paulo ; 41(4): 235-8, July-Aug. 1999.
Artículo en Inglés | LILACS | ID: lil-246832

RESUMEN

We studied 12 Hb C carriers: 4 homozygotic Hb CC and 8 heterozygotic. We observed the presence of free crystals in the peripheral blood of the homozygotes but in none of the heterozygotes. However, after incubation with 3 percent NaCl we were able to detect crystals in the heterozygotes (Hb AC and Hb SC), and in the homozygotes (Hb CC). In patient 04 (P04) less crystals formation occurred due to inhibition of the process by the presence of elevated levels of Hb F (12.2 percent). All the homozygotic patients had a splenomegaly of 3 to 6 fingerbreadths.We believe that the spleen wears off with time, thus allowing the passage of crystals to the peripheral blood. This finding might be associated with splenic insufficiency without a reduction of its dimensions. Finally, the finding of crystals in the peripheral blood permitted the diagnosis of Hb C obviating the need for electrophoresis


Asunto(s)
Humanos , Enfermedad de la Hemoglobina C/diagnóstico , Hemoglobina C/química , Esplenomegalia/sangre , Cristalización , Electroforesis en Gel de Agar , Homocigoto , Bazo/fisiopatología
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