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N Blin

Publications and source records attributed to N Blin.

At least 19 recordsLinked to original sources
PubMed2006

Two classes of outer hair cells along the tonotopic axis of the cochlea.

The molecular basis of high versus low frequency hearing loss and the differences in the sensitivity of outer hair cells depending on their cochlear localization are currently not understood. Here we demonstrate the existence of two different outer hair cell phenotypes along the cochlear axis. Outer hair cells in low frequency regions exhibit early sensitivity for loss of Ca(v)1.3 (alpha1 subunit 1.3 forming the class D L-type voltage-gated Ca(2+) channel), while high frequency regions display a progressive susceptibility for loss of the Ca(2+)-activated large conductance K(+) (BK) channel. Despite deafness, young Ca(v)1.3-deficient mice displayed distortion-product otoacoustic emissions (DPOAEs), indicating functional outer hair cells in the higher frequency range of the cochlea. Considering that DPOAEs are also found in the human deafness syndrome DFNB9 caused by mutations in the synaptic vesicle protein otoferlin, we tested the expression of otoferlin in outer hair cells. Surprisingly, otoferlin showed a distinct tonotopic expression pattern at both the mRNA and protein level. Otoferlin-expressing, Ca(v)1.3 deletion-sensitive outer hair cells in the low frequency range could be clearly separated from otoferlin-negative, BK deletion-sensitive outer hair cells in the high frequency range. In addition, BK deletion led to a higher noise vulnerability in low frequency regions, which are normally unaffected by the BK deletion alone, suggesting that BK currents are involved in survival mechanisms of outer hair cells under noise conditions. Our findings propose new mechanisms and candidate genes for explaining high and low frequency hearing loss.

Acoustic Stimulation↗
PubMed2006

C/EBPbeta is over-expressed in gastric carcinogenesis and is associated with COX-2 expression.

The CCAAT/enhancer-binding protein beta (C/EBPbeta) transcription factor has been associated with several cancer models. In this study, the expression of C/EBPbeta was analysed in a series of 90 gastric carcinomas (GCs). We also assessed the effect of C/EBPbeta on COX-2 expression. In normal gastric mucosa, C/EBPbeta expression was restricted to cells in the proliferative zone. In intestinal metaplasia, dysplasia, and GC of the intestinal and atypical subtypes, C/EBPbeta was over-expressed (p < 0.0001, for the association with histological type). C/EBPbeta and Ki67, a marker of cell proliferation, were also co-expressed in primary GC. We also observed an overlap between C/EBPbeta and COX-2 expression in GC. Using GC cell lines we show that C/EBPbeta can regulate the expression of endogenous COX-2 and transactivate the promoter of the COX-2 gene, depending on its methylation status. These results suggest that C/EBPbeta may be a marker of neoplastic transformation and also play an active role in gastric tumourigenesis by regulating COX-2 expression.

Adult↗
PubMed2005

Loss of function mutations of the GJB2 gene detected in patients with DFNB1-associated hearing impairment.

Mutations in GJB2, which encodes the gap junction protein connexin 26 (Cx26), are one of the major causes for inherited and sporadic nonsyndromic hearing impairment. This study aimed to functionally characterize more frequent GJB2 mutations identified in patients showing nonsyndromic hearing impairment. Following injection of wild type and mutated cRNA in Xenopus oocytes, Cx26 hemichannel activity was measured by depolarization activated conductance in noncoupled oocytes. All mutants showed a partially or completely defective phenotype, except (V27I)Cx26, a polymorphism tested as positive control. Coexpression of wild type and mutant Cx26 injected at equimolar levels revealed that p.M34T, p.V37I and p.I82M, but not p.G59V, p.L90P, p.R127H and p.R143W exert a dominant inhibitory effect. When coexpressed with Cx30, a connexin partially colocalized with Cx26 in the cochlea, all mutants had a dominant behavior. This study provides data that might be important for the improvement of genetic diagnosis and counseling for patients with hearing impairment.

Animals↗
PubMed2004

PCR-induced sequence alterations hamper the typing of prehistoric bone samples for diagnostic achondroplasia mutations.

Achondroplasia (ACH) is a skeletal disorder (MIM100800) with an autosomal dominant Mendelian inheritance and complete penetrance. Here we report the screening of ancient bone samples for diagnostic ACH mutations. The diagnostic G-->A transition in the FGFR3 gene at cDNA position 1138 was detected in cloned polymerase chain reaction (PCR) products obtained from the dry mummy of the Semerchet tomb, Egypt (first dynasty, approximately 4,890-5,050 BP [before present]), and from an individual from Kirchheim, Germany (Merovingian period, approximately 1,300-1,500 BP), both of which had short stature. However, these mutations were also reproducibly observed in four ancient control samples from phenotypically healthy individuals (false-positives), rendering the reliable molecular typing of ancient bones for ACH impossible. The treatment of a false-positive DNA extract with uracil N-glycosylase (UNG) to minimize type 2 transitions (G-->A/C-->T) did not reduce the frequency of the false-positive diagnostic ACH mutations. Recently, it was suggested that ancient DNA extracts may induce mutations under PCR. Contemporary human template DNA from a phenotypically healthy individual was therefore spiked with an ancient DNA extract from a cave bear. Again, sequences with the diagnostic G-->A transition in the FGFR3 gene were observed, and it is likely that the false-positive G-->A transitions result from errors introduced during the PCR reaction. Amplifications in the presence of MnCl(2) indicate that position 1138 of the FGFR3 gene is particularly sensitive for mutations. Our data are in line with previously published results on the occurrence of nonrandom mutations in PCR products of contemporary human mitochondrial HVRI template DNA spiked with ancient DNA extracts.

Achondroplasia↗
PubMed2004

Impairment of MLH1 and CDKN2A in oncogenesis of laryngeal cancer.

Our study aimed at elucidating which genetic alterations tend to form a network and could be applied as molecular markers of larynx squamous cell carcinoma (LSCC). A panel of genes involved in tumorigenesis was investigated. To search for the possible mechanisms of gene silencing, loss of heterozygosity (LOH) was analysed followed by testing DNA methylation and protein expression for those genes found with the highest frequency of LOH (CDKN2A (55.4%), MLH1 (46.0%), RB1 (35.7%)). A correlation of both LOH and hypermethylation with the loss of expression for CDKN2A and MLH1 was found. Disrupted Rb pathway (loss of expression of RB1 and/or of CDKN2A) in 55.9% of analysed cases confirmed the hypothesis that RB1 pathway is altered in head and neck squamous cell carcinomas, with CDKN2A (45%), rather than RB1 (11.8%) being more frequently inactivated. In LSCC, LOH tends to occur together in gene pairs or triplets. The pair MLH1/CDKN2A and triplets MLH1/TSG on 8p22/CDKN2A and MLH1/CDKN2A/RB1 are related to staging and grading. LOH in MLH1 correlates with lower and LOH in CDKN2A with higher grades of LSCC. It can be concluded that MLH1 and CDKN2A play an important role in LSCC development and progression.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Refinement of the DFNA4 locus to a 1.44 Mb region in 19q13.33.

Many forms of autosomal dominant non-syndromic hearing impairment are known. While the underlying gene defects and causative mutations have been discovered for some forms, the gene responsible for DFNA4 has remained elusive to date. Examination of a German four-generation kindred led to the identification of a 1.44 Mb map segment in contig NT_011109 as being the most likely DFNA4 candidate region in 19q13.33. The recombination breakpoints in this family and the intervals of two previously reported DFNA4 families allowed us to delineate a minimum consensus region between the markers D19S879 and D19S246. In our family, a maximum two-point LOD score of 4.5 was obtained at theta = 0 for the marker D19S867. Within the refined DFNA4 interval the public databases list more than 50 genes, from which several appear to be promising DFNA4 candidates due to similarities with animal models and with other causative genes involved in hearing disability.

Adult↗
PubMed2004

[Mitochondrial hearing impairment. Background, genetic predisposition and possibilities for diagnosis].

Hearing impairment (HI) is one of the most common neurosensory disorders, with sensorineural hereditary HI being the most common form. Mitochondrial maternally inherited HI appears to be increasing in frequency. The incidence of mitochondrial defects causing HI is estimated to be between 6 and 33% of all hearing deficiencies, with an even higher percentage for some syndromic cases. This review summarises the syndromic and non-syndromic characteristics of sensorineural HI based on mutations in mitochondrially encoded genes, the relationship to aminoglycoside-induced HI and related diagnostic tools.

DNA, Mitochondrial↗
PubMed2004

Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation.

BACKGROUND: Low RNA yields from clinical samples are a limiting step for microarray technology. AIMS: To design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the crucial step of global mRNA amplification performed before microarray hybridisation, using less than 1 microg total RNA. METHODS: Three RNA extraction procedures were compared for small size samples. Total RNA was amplified from universal RNA or the BC-H1 breast cancer micrometastatic cell line using three different protocols. Real time quantitative PCR technology was used for accurate measurement of urokinase plasminogen activator receptor and cytokeratin 8 RNA amplification rates and ratios, using primer sets binding at various distances from the 3' end of transcripts. A 50 mer oligomeric array targeting 87 genes potentially involved in breast cancer metastatic progression was built and hybridised with amplified RNA. RESULTS: Eighteen nanograms of total RNA could be purified from 1000 BC-H1 micrometastatic cells. Amplification rates of 25,000 to 100,000 were achieved with as little as 10 ng of starting material. However, results were highly variable, depending on the amount of starting material, gene characteristics, sample quality, and protocols used. Oligomeric array hybridisation with 20 microg reference RNA resulted in specific and reproducible signals for 83% of the genes, whereas mRNA amplification from less than 400 ng of starting material resulted in selective detection of signals from highly expressed genes. CONCLUSIONS: Improvements in the design of global mRNA amplification procedures and oligomeric arrays are needed to extract informative gene expression data from clinical samples containing limited cell numbers.

Base Sequence↗
PubMed2003

[Clinical and molecular genetic analysis of monozygotic twins displaying stapes gusher syndrome (DFN3)].

BACKGROUND: DFN3 ( "stapes gusher") is the most frequent form of X-linked hearing impairment. It accounts for up to 0.5% of all cases of severe childhood hearing disorders. PATIENTS AND METHODS: Monozygotic twins with suspected stapes gusher syndrome, their mother, and control individuals were analyzed clinically and genetically. RESULTS: The clinical investigations confirmed a DFN3 phenotype in both brothers who displayed all typical symptoms. A molecular genetic investigation of the POU3F4 gene, which plays an essential role in the development of DFN3, was also performed. No chromosomal aberrations within the coding region of POU3F4were detected. Since several authors have described mutations in the 5' untranslated region of the gene also resulting in a DFN3 phenotype, we screened this area by microsatellite analysis and detected a double deletion localized in the critical interval. This is the first description of a double deletion in the non-coding region of POU3F4 leading to DFN3 phenotype. CONCLUSION: Interestingly, in spite of having an identical genotype, the twins displayed significant phenotypic differences. This underlines the importance of exogenous factors in the development of inherited pathological processes.

Audiometry, Pure-Tone↗
PubMed2003

[Phenotype of patients showing hearing impairment based on the 35delG mutation in the connexin 26 gene].

BACKGROUND: Hereditary hearing impairment constitutes a heterogeneous class of disorders showing different patterns of inheritance and involving multiple genes. Mutations in the GJB2 gene, especially the 35delG mutation, have been established as a major cause of inherited and sporadic nonsyndromic hearing impairment in different populations. METHODS: We analyzed 14 northeast Hungarian families and 69 sporadic cases with nonsyndromic hearing impairment for the 35delG mutation. Sixty-five patients showing a homozygous 35delG mutation were examined regarding their audiologic phenotype. RESULTS: In general, these patients (70%) showed a prelingual, sensorineural, bilateral, symmetric hearing impairment without progression. The audiograms demonstrated sloping as well as flat patterns. CONCLUSIONS: The severity of hearing impairment varied in 30% of all analyzed patients, making genetic counseling difficult.

Acoustic Impedance Tests↗
PubMed2003

Tumour necrosis factor alpha and nuclear factor kappaB inhibit transcription of human TFF3 encoding a gastrointestinal healing peptide.

BACKGROUND: and aims: Tumour necrosis factor alpha (TNF-alpha) induction of nuclear factor kappaB (NFkappaB) activation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Trefoil factor family peptides TFF1, TFF2, and TFF3 exert protective, curative, and tumour suppressive functions in the gastrointestinal tract. In this study, we investigated effects of the TNF-alpha/NFkappaB regulatory pathway by TNF-alpha on expression of TFFs. METHODS: After TNF-alpha stimulation, expression of TFF genes was analysed by quantitative real time polymerase chain reaction and by reporter gene assays in the gastrointestinal tumour cell lines HT-29 and KATO III. Additionally, NFkappaB subunits and a constitutive repressive form of inhibitory factor kappaB (IkappaB) were transiently coexpressed. In vivo, morphological changes and expression of TFF3, mucins, and NFkappaB were monitored by immunohistochemistry in a rat model of 2,4,6-trinitrobenzene sulphonic acid induced colitis. RESULTS: TNF-alpha stimulation evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Downregulation of reporter gene transcription of TFF3 was observed with both TNF-alpha and NFkappaB, and was reversible by IkappaB. In vivo, the increase in epithelial expression of NFkappaB coincided with reduced TFF3 expression during the acute phase of experimental colitis. CONCLUSIONS: Downregulation of intestinal trefoil factor TFF3 is caused by repression of transcription through TNF-alpha and NFkappaB activation in vitro. In IBD, perpetual activation of NFkappaB activity may contribute to ulceration and decreased wound healing through reduced TFF3.

Animals↗
PubMed2002

Substitutions in the conserved C2C domain of otoferlin cause DFNB9, a form of nonsyndromic autosomal recessive deafness.

DFNB, the nonsyndromic hearing loss with an autosomal recessive mode of inheritance constitutes the majority of severe to profound prelingual forms of hearing impairment, usually leading to inability of speech acquisition. We analyzed a consanguineous family with autosomal recessive deafness which has been shown to segregate within chromosomal region 2p23.1 (DFNB9; MIM 601071). By SSCP analysis and DNA sequencing of the 48 exons of the DFNB9 gene, coding for otoferlin, previously reported mutations in OTOF were excluded. Next to a frequent T > C single nucleotide polymorphism in exon 8, two novel mutations linked in exon 15 of the OTOF long splice form were identified comprising substitutions at positions 490 (Pro > Gln) and 515 (Ile > Thr), both located in the conserved Ca(2+) binding C2C domain of this peptide. Comparisons of homology using human and mice otoferlins and closely related peptides and computer simulation analyses suggest that changes in the mutated segment's secondary structure affect the Ca(2+) binding capacity of the C2C domain in otoferlin.

Alternative Splicing↗
PubMed2002

Gastroprotective peptide trefoil factor family 2 gene is activated by upstream stimulating factor but not by c-Myc in gastrointestinal cancer cells.

BACKGROUND: Damage to the gastrointestinal mucosa results in the acute up-regulation of the trefoil factor family peptides TFF1, TFF2, and TFF3. They possess protective, healing, and tumour suppressive functions. Little is known about the regulation of TFF gene expression. The promoters of all three TFF genes contain binding sites (E box) for upstream stimulating factor (USF) and Myc/Max/Mad network proteins. AIMS: To determine the nature and function of transcription factors that bind to these E boxes and to understand their role for TFF gene expression. METHODS: TFF promoter activities were determined by reporter gene assays. DNA binding was monitored by electromobility shift assays and by chromatin immunoprecipitation analyses. Expression of endogenous TFF was determined by multiplex RT-PCR. RESULTS: It was observed that the TFF2 promoter is specifically and efficiently activated by USF transcription factors but not by c-Myc. USF displayed comparable binding to a high affinity Myc/Max binding site compared with the three TFF E boxes, while c-Myc exhibited lower affinity to the TFF E boxes. In contrast, pronounced binding differences were observed in cells with a strong preference for USF to interact specifically with the TFF2 E box, while Myc was not above background. Exogenous expression of USF was sufficient to activate the chromosomal TFF2 and to a lesser extent, the TFF1 gene. CONCLUSION: These findings define USF factors as regulators of the TFF2 gene and suggest that promoter specific effects are important for a pronounced gene activation of this cytoprotective peptide.

Binding Sites↗
PubMed2001

Dermcidin: a novel human antibiotic peptide secreted by sweat glands.

Antimicrobial peptides are an important component of the innate response in many species. Here we describe the isolation of the gene Dermcidin, which encodes an antimicrobial peptide that has a broad spectrum of activity and no homology to other known antimicrobial peptides. This protein was specifically and constitutively expressed in the sweat glands, secreted into the sweat and transported to the epidermal surface. In sweat, a proteolytically processed 47-amino acid peptide was generated that showed antimicrobial activity in response to a variety of pathogenic microorganisms. The activity of the peptide was maintained over a broad pH range and in high salt concentrations that resembled the conditions in human sweat. This indicated that sweat plays a role in the regulation of human skin flora through the presence of an antimicrobial peptide. This peptide may help limit infection by potential pathogens in the first few hours following bacterial colonization.

Amino Acid Sequence↗
PubMed2001

Microsatellite and chromosome instability in squamous cell laryngeal carcinoma.

Head-and neck squamous cell carcinoma (HNSCC) represents almost 5% of all malignancies in Europe. The aetiology of HNSCC is complex, with both genetic and mutagenic factors involved. The aim of the present study was to investigate the loss of heterozygosity (LOH), mainly at tumour suppressor loci (using markers D1S2883, D2S123, D3S1611, D5S346, D7S501, D8S254, TP53, NM23), microsatellite instability (BAT25, 26, 40) and (bleomycin test) in patients with squamous cell larynx cancer. In a group of 20 patients LOH was observed mainly at the loci 3p (64.7%), 8q (71.4%), 17q (M1-30.8%, M2-25%, M3-38.5%). Despite chromosomal instability detected by bleomycin no microsatellite instability was observed.

Adaptor Proteins, Signal Transducing↗
PubMed2001

[An attempt to identify the most frequent genomic mutations responsible for isolated deafness in patients after cochlear implantation].

The aim of this study was to identify subjects with 35delG mutation of GJB2 gene as the most frequent genetic cause of deafness. Deaf patients receiving cochlear implantation at the ENT Clinic at University of Medical Sciences in Poznań and their family members were recruited to the study. Peripheral blood lymphocytes DNA was amplified in allele-specific PCR and analysed for single strand conformation polymorphism (SSCP) to detect mutation at DFNB1 locus. 35delG mutation at both alleles was found at 42.9% of deaf patients and 29.4% of health relatives were found to be carrier of the mutation at one allele. The study is thought to be a first step in analysis of typical mutations in Polish deaf population.

Adolescent↗