Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
Add more filters










Publication year range
1.
Braz J Med Biol Res ; 54(7): e10579, 2021.
Article in English | MEDLINE | ID: mdl-34008754

ABSTRACT

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.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Cochlea , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation , Hair Cells, Auditory , Mice , Organ of Corti , Receptors, Notch , Transcription Factor HES-1/genetics
2.
Braz. j. med. biol. res ; 54(7): e10579, 2021. tab, graf
Article in English | LILACS | ID: biblio-1249313

ABSTRACT

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.


Subject(s)
Animals , Rats , Cochlea , Basic Helix-Loop-Helix Transcription Factors/genetics , Organ of Corti , Cell Differentiation , Receptors, Notch , Transcription Factor HES-1/genetics , Hair Cells, Auditory
3.
Braz J Med Biol Res ; 49(4): e5064, 2016.
Article in English | MEDLINE | ID: mdl-27007652

ABSTRACT

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350 g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.


Subject(s)
Hair Cells, Auditory, Inner/transplantation , Hearing Loss, Sensorineural/surgery , Organ of Corti/surgery , Stem Cell Transplantation/methods , Stem Cells , Animals , Auditory Threshold , Cell Survival , Cells, Cultured , Evoked Potentials, Auditory, Brain Stem , Female , Guinea Pigs , Immunohistochemistry , Mice, Inbred BALB C , Neomycin , Protein Synthesis Inhibitors , Reproducibility of Results , Treatment Outcome
4.
Braz. j. med. biol. res ; 49(4): e5064, 2016. tab, graf
Article in English | LILACS | ID: biblio-951670

ABSTRACT

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.


Subject(s)
Animals , Female , Organ of Corti/surgery , Stem Cells , Stem Cell Transplantation/methods , Hair Cells, Auditory, Inner/transplantation , Hearing Loss, Sensorineural/surgery , Auditory Threshold , Immunohistochemistry , Protein Synthesis Inhibitors , Neomycin , Cell Survival , Cells, Cultured , Reproducibility of Results , Evoked Potentials, Auditory, Brain Stem , Treatment Outcome , Guinea Pigs , Mice, Inbred BALB C
5.
Acta Neuropathol ; 101(3): 202-10, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11307618

ABSTRACT

Tuberous sclerosis complex is an autosomal dominant multisystem disorder, characterized by the development of hamartomas in multiple organs, primarily the skin, heart, kidney, and brain. The tuberous sclerosis genes, TSC1 and TSC2, encode hamartin and tuberin, respectively. Employing specific antibodies for hamartin and tuberin, we analyzed the expression of these two proteins by Western blot analyses in normal developing human and rat tissues. Both proteins are expressed ubiquitously in human fetal tissues and placenta, but are expressed at relatively low levels in human adult tissues, except brain. Similarly, high expression of these two proteins is observed in rat embryonic tissues, with a progressive decline after birth. To better characterize the developmental expression of tuberin and hamartin, we conducted a detailed study in rat tissues from embryonic day 13 to adult by Western blot analysis and immunohistochemistry. Immunohistochemical staining of rat tissues for tuberin and hamartin revealed tissue-specific expression patterns throughout development. Both tuberin and hamartin are expressed in epithelia, muscle (smooth, cardiac and skeletal muscle) and the nervous system (neurons, glia, choroid plexus and arachnoid). Except for the central nervous system, immunostaining intensity declines with age, confirming the protein blot analysis. These results indicate that tuberin and hamartin may play a critical role in development, and thus provide a framework for understanding the developmental and hamartomatous manifestations of tuberous sclerosis. These findings also suggest that tuberin and hamartin have additional functions in the adult brain, consistent with the marked neurological problems that afflict many patients with tuberous sclerosis.


Subject(s)
Gene Expression Regulation, Developmental , Proteins/metabolism , Repressor Proteins/metabolism , Tuberous Sclerosis/metabolism , Age Factors , Animals , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Epithelium/metabolism , Epithelium/ultrastructure , Female , Fetus/metabolism , Fetus/pathology , Humans , Immunohistochemistry , Pregnancy , Rats , Rats, Wistar , Tuberous Sclerosis/pathology , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins , Viscera/metabolism , Viscera/pathology
6.
Am J Physiol Renal Physiol ; 278(5): F737-46, 2000 May.
Article in English | MEDLINE | ID: mdl-10807585

ABSTRACT

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in multiple organs, notably the brain and kidneys. The disease is caused by mutations in TSC1 or TSC2 genes, coding hamartin and tuberin, respectively. Immunofluorescence analysis of tuberin and hamartin performed here demonstrates that both proteins are specifically expressed in the distal urinary tubule, comprising the distal tubules, connecting segment, and collecting ducts. Hamartin, distinct from tuberin, is expressed in the thick ascending limbs of Henle and in juxtaglomerular cells, where it colocalizes with renin. In positive epithelial cells, tuberin localizes to the cytoplasm as well as the apical membrane. Hamartin, however, preferentially localizes to the apical membrane. The two proteins colocalize at the apical membrane of type A intercalated cells and connecting tubule cells, whereas in type B intercalated cells they reveal a variable pattern of expression. The cell-specific expression of tuberin and hamartin described here will provide critical insight into the cell types that give rise to kidney lesions, and the tumor suppressor role of these proteins in TSC.


Subject(s)
Kidney/metabolism , Proteins/metabolism , Repressor Proteins/metabolism , 3T3 Cells , Animals , Antibodies, Monoclonal , COS Cells , Cell Line , HeLa Cells , Humans , Kidney Tubules/metabolism , Kidney Tubules, Collecting/metabolism , Mice , Mutation , Proteins/genetics , Proteins/immunology , Rats , Rats, Sprague-Dawley , Repressor Proteins/genetics , Repressor Proteins/immunology , Subcellular Fractions/metabolism , Tissue Distribution , Tuberous Sclerosis/genetics , Tuberous Sclerosis/metabolism , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins
7.
Hum Mol Genet ; 9(3): 347-52, 2000 Feb 12.
Article in English | MEDLINE | ID: mdl-10655543

ABSTRACT

We screened 42 sporadic gastric tumors and found that seven of them had significant microsatellite instability. These were then studied at 26 microsatellite loci, comprising di-, tri- and tetranucleotide repeats. The instability level of individual microsatellites in the tumors was found to be positively correlated with the population average heterozygosity and variance of repeat number of the microsatellite loci, as predicted by the stepwise mutation model. Moreover, as is known to occur in human populations, instability was strongly correlated with the number of repeats at each microsatellite locus and with the perfection of the reiterated sequence. These results demonstrate that microsatellite mutations in unstable tumors show similarities to germline mutations and suggest that their study may be useful in understanding the mechanisms that generate microsatellite variability in human populations. We used this model to test the claims that the microsatellite mutation process is biased towards increased size and heterozygosity with wide differences in allele sizes. These assertions were not confirmed.


Subject(s)
Adenocarcinoma/genetics , Gastrointestinal Neoplasms/genetics , Microsatellite Repeats , DNA, Neoplasm/analysis , Female , Humans , Male , Models, Genetic , Mutagenesis
8.
Am J Med Genet ; 84(3): 198-201, 1999 May 28.
Article in English | MEDLINE | ID: mdl-10331590

ABSTRACT

We used a non-isotopic polymerase chain reaction (PCR) technique for fragile X syndrome diagnosis to screen 256 mentally retarded boys who were selected randomly from special schools. Patients identified as pre- or full-mutation carriers were further investigated by Southern blot analysis with the StB12.3 probe. The PCR-based test identified five boys with the expanded allele and 17 other patients as carriers of either premutated or gray-zone alleles. The full mutation was confirmed in four cases after Southern blotting and a fifth patient carried a normal allele. Of the 17 patients identified with a premutation allele by PCR, one individual was diagnosed as mosaic by Southern blotting, 12 individuals displayed fragments of 2.90 kb or 2.85 kb, and the remaining four individuals showed apparently normal-sized fragments. However, sizing of these 16 alleles by further PCR analysis showed them to be in the gray-zone range (40-60 repeats). Therefore, the frequency of the full mutation in this cohort of mentally retarded boys was close to 2% (5/256). The prevalence of gray-zone alleles among those mentally impaired boys who did not carry the full mutation was 6.4% (16/251) and, although more than twice the prevalence of these alleles among a cohort of unaffected Brazilian males 2.8% (71251), the difference did not reach statistical significance.


Subject(s)
Alleles , Fragile X Syndrome/genetics , Intellectual Disability/genetics , Brazil , DNA/analysis , Humans , Male , Mutation , Polymerase Chain Reaction
9.
Gene ; 223(1-2): 369-74, 1998 Nov 26.
Article in English | MEDLINE | ID: mdl-9858768

ABSTRACT

Recently, several important human genetic diseases have been shown to be due to pathological expansion of expressed trinucleotide microsatellites. Discovery of other such 'expansion diseases' will depend on characterization of more expressed loci containing trinucleotide repeats. We searched the expressed sequence tag database (dbEST) for repetitive trinucleotides and selected four loci for further studies (EST00586 EST05486, EST13299 and HHEA48B). For each locus, we have identified size polymorphism by PCR amplification and achieved chromosomal mapping using a somatic hybrid cell panel and the Stanford G3 radiation hybrid panel. Further searches of GenBank, dbEST and UniGene unraveled EST clusters for three of the loci, allowing construction of contigs and, in one instance, identification of a partial open reading frame. Three of the loci were linked to autosomal dominant human genetic diseases whose primary gene defect has not yet been established. Although it is a priori improbable that there is an etiological connection between the loci studied and the diseases, our results demonstrate that dbEST constitutes a useful starting point in the search for candidate loci for new expansion diseases.


Subject(s)
Chromosome Mapping/methods , Expressed Sequence Tags , Polymorphism, Genetic , Trinucleotide Repeats , Chromosomes, Human , Databases, Factual , Humans
10.
Hum Genet ; 102(2): 231-5, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9521596

ABSTRACT

Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)3CAC(CAT)CAC(CAT)2CAC(CAT)CAC(CAT)15, thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms.


Subject(s)
Mutation , Neoplasms/genetics , Trinucleotide Repeats , Adenocarcinoma/genetics , Cloning, Molecular , DNA, Bacterial/analysis , Genetic Markers , Humans , Polymerase Chain Reaction , Recombinant Proteins/genetics , Sequence Analysis, DNA , Stomach Neoplasms/genetics
11.
Braz J Med Biol Res ; 30(8): 915-21, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9361718

ABSTRACT

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in tumor suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra- and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70% of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing.


Subject(s)
Colorectal Neoplasms/genetics , Microsatellite Repeats/genetics , Brazil , Humans
12.
Braz. j. med. biol. res ; 30(8): 915-21, Aug. 1997. ilus, tab
Article in English | LILACS | ID: lil-197245

ABSTRACT

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra-and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70 per cent of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing.


Subject(s)
Humans , Colorectal Neoplasms/genetics , Microsatellite Repeats/genetics , Brazil , Genes, DCC , Polymerase Chain Reaction
13.
Hum Genet ; 99(6): 796-800, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9187676

ABSTRACT

Long expansions of transcribed trinucleotide microsatellites have been etiologically associated with some neurological diseases. The investigation of such novel polymorphisms has thus become a subject of great interest. We searched the expressed sequence tag databank for reiterated trinucleotides and selected EST00493 (D2S196E) with 14 tandem ACA triplets as a potentially polymorphic locus. Size variation was readily detected, with four common alleles containing 12-15 repeats. In addition, we observed distinct heteroduplexes in amplifications from individuals with identical ACA genotypes. Sequencing of their polymerase chain reaction (PCR) products revealed a G-->A transition immediately preceding the trinucleotide repeats, hence defining 8 distinct haplotypes and 36 possible genotypes. Indeed, mutation detection enhancement gel electrophoresis of mixed PCR products from cloned haplotypes revealed 24 distinct heteroduplex patterns for the six possible trinucleotide heterozygotes. The observation of heteroduplex patterns in non-denaturing polyacrylamide gel electrophoresis (instead of the more commonly used denaturing gels) can thus be utilized to increase the informativeness of microsatellite polymorphisms by unraveling otherwise cryptic sequence variation. The D2S196E polymorphism has proved useful for demonstrating microsatellite instability and loss of heterozygosity in colorectal tumors.


Subject(s)
Polymorphism, Genetic , Transcription, Genetic , Trinucleotide Repeats/genetics , Base Sequence , Colorectal Neoplasms/genetics , Electrophoresis, Polyacrylamide Gel , Haploidy , Humans , Microsatellite Repeats , Molecular Sequence Data , Nucleic Acid Heteroduplexes/genetics , Phenotype , Polymerase Chain Reaction
15.
Am J Med Genet ; 64(2): 270-3, 1996 Aug 09.
Article in English | MEDLINE | ID: mdl-8844063

ABSTRACT

The size of the CGG repeat of the FMR1 gene was investigated with probe StB12.3 in 154 transmissions to the offspring of heterozygotes for the premutation and the full mutation. Among the 135 offspring of premutated heterozygotes there were three decreases in size of the repeats: in two of these cases a full mutation was present along with the decreased premutation, and in a third mosaic (46,fra(X)(q27.3),Y), a normal allele was observed. In the 19 offspring of fully mutated females with no detected mosaicism, there were three mosaics and three individuals who had full mutations that included a number of repeats smaller than those present in their mothers. Among the 32 offspring who received a premutation from their premutated mothers, 27 alleles were increased in size and 5 remained unaltered. Among 11 mosaic offspring of premutated mothers, the premutation increased in 4, decreased in 3, and was unchanged in 4. In contrast to the trend of an increasing premutation size in the non-mosaic offspring, the premutation present in mosaics can be smaller, larger, or of unaltered size with approximately equal frequencies. These data suggest that the premutations present in mosaics result from mitotic instability of the inherited full mutations. This is further supported by the finding of a mosaic male with a normal sized allele.


Subject(s)
Fragile X Syndrome/genetics , Heterozygote , Mosaicism , Mutation , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Trinucleotide Repeats , DNA/blood , Female , Fragile X Mental Retardation Protein , Genomic Imprinting , Humans , Male , Restriction Mapping
16.
Hum Genet ; 97(6): 808-12, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8641701

ABSTRACT

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)29 allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.


Subject(s)
Fragile X Syndrome/diagnosis , Fragile X Syndrome/genetics , Polymerase Chain Reaction/methods , RNA-Binding Proteins , Alleles , Base Composition , Base Sequence , Blotting, Southern/methods , Dinucleotide Repeats/genetics , Female , Fragile X Mental Retardation Protein , Genetic Carrier Screening , Genetic Testing , Humans , Intellectual Disability/genetics , Male , Molecular Sequence Data , Mosaicism , Nerve Tissue Proteins/genetics , Sensitivity and Specificity , Trinucleotide Repeats/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...