1,721,093 research outputs found
PHF5A represents a bridge protein between splicing proteins and ATP-dependent helicases and is differentially expressed during mouse spermatogenesis
No full textPHF5A is a highly conserved protein from yeast to man, and based on studies in yeast, it was suggested that the homologous protein RDS3P in S. cerevisiae takes part in the organization of U2 snRNP particles. By using the yeast two-hybrid assay we could demonstrate that PHF5A interacted both with ATP-dependent helicases EP400 and DDX1 and with arginine-serine (RS)-rich domains of splicing factors U2AF1 and SFRS5 in mouse. Furthermore, domain interaction studies revealed that PHF5A interaction with EP400 and DDX1 is restricted to the N-terminal part of PHF5A, whereas the C-terminal region of PHF5A was found to be responsible for the association with U2AF1 and SFRS5. By using the yeast three-hybrid assay, we could further show that both EP400 and DDX1 interacted only indirectly with U2AF1 and SFRS5 proteins via the bridge protein PHF5A. The subcellular localization of a PHF5A-GFP fusion protein was predominantly observed in the nucleus and, in addition, PHF5A co-localized with both U2AF1 and SFRS5 proteins in nuclear speckles of NIH3T3 cells. Moreover, expression analyses demonstrated that PHF5A and U2AF1 gene expression coincided in spermatocytes during murine spermatogenesis and interaction between these proteins was also detectable in the spermatocyte-specific cell line GC-4spc by using in vivo co-immunoprecipitation studies. Taken together, our results indicate that PHF5A resembles a protein which interacts with splicing factors U2AF1 and SFRS5 and helicases EP400 and DDX1 and functions as a bridge protein between these proteins. Copyright (c) 2008 S. Karger AG, Basel.Deutsche Forschungsgemeinschaft [SFB 271
A Novel MECP2 Mutation in a Boy with Neonatal Encephalopathy and Facial Dysmorphism
No full textMethly-CpG-binding protein 2 (MECP2) mutations cause Rett syndrome in females. Here we report on a male infant with neonatal encephalopathy, myoclonic jerks, and irregular breathing patterns caused by a novel frameshift mutation in the MECP2 gene. In addition he has facial dysmorphisms previously not described in these patients. (J Pediatr 2009; 155:140-3
Factor XII deficiency is strongly associated with primary recurrent abortions
No full textObjective: To evaluate factor XII deficiency in women with primary and secondary recurrent abortion. Design: Prospective case-control study. Setting: University hospital. Patient(s): Sixty-seven women with primary and 33 women with secondary recurrent abortion of unexplained nature and 49 healthy controls with no history of thrombotic disease or adverse pregnancy outcomes. Main Outcome Measure(s): Plasma factor XII activity, activated protein C resistance, factor V Leiden mutation analysis, protein C, protein S, antithrombin III, karyotyping, and anticardiolipin antibodies. Result(s): Ten of 67 women with primary recurrent abortion (14.9%) and 4 of 33 women (12.1%) with secondary recurrent abortion had reduced factor XII activity (<60%). These results are highly significant in the former group and showed a tendency toward significance in the latter group. All controls had normal factor XII activity. Conclusion(s): Factor XII deficiency is strongly associated with primary recurrent abortion, and women with secondary recurrent abortion show a tendency toward factor XII deficiency. (C) 2003 by American Society for Reproductive Medicine
Dual silencing of insulin-like growth factor-I receptor and epidermal growth factor receptor in colorectal cancer cells is associated with decreased proliferation and enhanced apoptosis
No full textOverexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal-regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-1-dependent phosphorylation of AKT but had no effect on IGF-1- or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy. [Mol Cancer Ther 2009;8(4): 821-33
Blockade of the type I IGF receptor expression in human prostate cancer cells inhibits proliferation and invasion, up‐regulates IGF binding protein‐3, and suppresses MMP‐2 expression
No full textThe type I insulin-like growth factor receptor (IGF-IR) is involved in tumour cell proliferation, invasion, and cancer cell survival. Several studies indicate that the IGF axis contributes to prostate cancer pathogenesis, but there is no consensus regarding the relative expression of the IGF-IR in benign and malignant prostate epithelium. In this study, endogenous IGF-IR gene expression was reduced in stably transfected PC-3 cells by employing an antisense RNA strategy which resulted in significant suppression of both PC3 cell invasion and proliferation in vitro. Furthermore, it was demonstrated that a direct correlation exists between the inhibition of IGF-IR gene expression and either up-regulation of IGF binding protein (BP)-3 or down-regulation of matrix metalloproteinase (MMP)2 expression in androgen-independent PC-3 cells. Moreover, inhibition of IGF-IR gene expression in transfected PC-3 cells leads to an enhanced rate of spontaneous apoptosis. In addition, expression analyses by quantitative RT-PCR on RNA from laser microdissected matched normal prostate and prostate tumour samples revealed that IGF-IR gene expression was up-regulated in nine of 12 prostate cancers, whereas IGFBP-3 gene expression was downregulated in all 12 prostate carcinomas analysed. These results indicate an important role for IGF-IR and IGFBP-3 in the homeostasis of prostate carcinoma cells and provide a further basis for targeting IGF-IR or IGFBP-3 gene expression in order to improve understanding of the IGF-IR-activated signalling pathways and as a potential treatment for prostate cancer. Copyright (C) 2003 John Wiley Sons, Ltd
Discordant phenotype in monozygotic twins with mosaic trisomy 12p in lymphocytes
No full textWe report on monochorionic diamniotic male twins discordant for the trisomy 12p syndrome. Trisomy 12p mosaicism with a supernumerary der(12)(pter > q12) was detected in approximately 50% of lymphocytes in both children. Fluorescence in situ hybridisation (FISH) revealed a high grade mosaicism of approximately 77% trisomy 12p cells in buccal smear and 85% in hair follicles in the affected twin, while in the normal developing brother an additional 12p chromosome fragment could not be detected in those tissues. Instead, in 3% of buccal smear and hair follicle cells a minute supernumerary marker chromosome comprising central portions of chromosome 12 was observed. Trisomy 12p mosaicism, confined to the lymphocytes of the unaffected twin, may be due to prenatal twin-to-twin transfusion, explaining the conspicuously discordant clinical phenotype. We discuss the possible sequence of events leading to the cytogenetic findings and compare the clinical phenotype presented in the affected twin with other cases of trisomy 12p and tetrasomy 12p (Pallister-Killian syndrome). (c) 2012 Elsevier Masson SAS. All rights reserved
Assessment of promoter elements of the germ cell-specific proacrosin gene
No full textThe testis-specific proacrosin gene encodes for a fertilization-promoting protein. In mouse and rat it is first transcribed in late pachytene spermatocytes and revealed to be translationally regulated. Former proacrosin promoter studies demonstrated that elements necessary for conducting a stage and temporal-specific expression of the gene are located within 0.9 kb upstream of the translational start codon. in the present study we analyzed putative cis-acting elements located in this promoter region for their specific binding properties to nuclear factors assumed to be involved in proacrosin gene regulation. Supplement of specific antibodies in electrophoretic mobility shift assays (EMSA) revealed that two Y-box proteins and the transcription factors CREM and YY1 interact with proacrosin promoter elements. The Y-box proteins, antigenically related to the frog Y-box proteins FRGY1 and FRGY2, bound to the Y-box (55-66 bp upstream of the ATG initiation codon) in brain and testis nuclear extracts, respectively. CREM bound to three elements (30-37, 252-259, and 717-724 bp upstream of ATG). The ubiquitous transcription factor YY1 bound to a conserved element in the central proacrosin promoter (457-473 bp upstream of ATG) and showed almost germ cell-specific truncates in EMSA. These results suggest that the identified factors are involved in proacrosin gene regulation. J. Cell. Biochem. 83: 155-162, 2001. (C) 2001 Wiley-Liss, Inc
A disease causing deletion of 29 base pairs in intron 15 in the MKS1 gene is highly associated with the campomelic variant of the Meckel-Gruber syndrome
No full textMeckel-Gruber syndrome (MKS) is an autosomal recessive disorder causing severe defects in the developing central nervous system and other organs. Recently, mutations in the MKS1 gene have been identified as disease causing in individuals of Finnish MKS families. The primary aim of the present study was to assess the frequency of the 'Finnish founder mutation' (29 bp IVS15-7_35) in the MKS1 gene in 20 aborted fetuses with a diagnosis of MKS. The secondary aim was to screen for novel mutations in the coding sequence of the MKS1 gene of MKS fetuses and to obtain genotype-phenotype correlations where possible. Furthermore, we evaluated the carrier rate of a deletion of 29 bp in intron 15 of the MKS1 gene in a German population. To identify and characterize mutations in the MKS1 gene, sequence analyses and quantitative real time polymerase chain reaction studies were performed. We could identify the same type of mutation, a deletion of 29 bp in intron 15 of the MKS1 gene, in 8 out of the 20 cases studied. Six out of the eight cases with such a mutation displayed the campomelic variant of MKS. The carrier frequency among 519 healthy German individuals was 1:260. This deletion in the MKS1 gene is highly associated with a distinct subtype of the MKS, namely the campomelic variant. In individuals of European origin suffering from the campomelic MKS variant, the described deletion is highly likely to be causative. Regarding the results of our study, the incidence of MKS in Germany can be estimated as 1:135,000. In families with a known mutation in the MKS1 gene, it is now possible to offer an early prenatal testing, for example with chorionic villus sampling and mutation analysis
cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections
No full textLaser microdissection is a valuable tool to prepare pure cell populations from complex tissues for further analyses. Gene expression studies by real-time RT-PCR and cDNA arrays of microdissected tissues are becoming widely used methods. The integrity and quantity of prepared RNA must be proven to ensure reliable results in subsequent applications such as quantitative RT-PCR and cDNA-arrays. In the present study we used RNAlater(TM) protected prostate tissue for laser microdissection of tumor and tumor-free tissues. RNA quality and quantity was assessed using automated capillary gel electrophoresis. By using quantitative real time-RT-PCR before and after mRNA amplification the insulin-like growth factor binding protein-3 (IGFBP-3) gene expression was shown to be down-regulated in three out of five cases and DD3 was up-regulated in cancer tissues in all cases. The up-regulation of DD3 and the down-regulation of IGFBP-3 gene expression in cancer tissue were conserved after RNA amplification. A cDNA microarray also revealed an IGFBP-3 down-regulation in cancer tissue as well as several genes known to be differerentially expressed in prostate cancer. Taken together, we present a novel method for the isolation of intact RNA from laser microdissection-derived prostate cancer tissue useful for downstream applications of real-time RT-PCR and cDNA microarrays
cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections
No full textLaser microdissection is a valuable tool to prepare pure cell populations from complex tissues for further analyses. Gene expression studies by real-time RT-PCR and cDNA arrays of microdissected tissues are becoming widely used methods. The integrity and quantity of prepared RNA must be proven to ensure reliable results in subsequent applications such as quantitative RT-PCR and cDNA-arrays. In the present study we used RNAlater(TM) protected prostate tissue for laser microdissection of tumor and tumor-free tissues. RNA quality and quantity was assessed using automated capillary gel electrophoresis. By using quantitative real time-RT-PCR before and after mRNA amplification the insulin-like growth factor binding protein-3 (IGFBP-3) gene expression was shown to be down-regulated in three out of five cases and DD3 was up-regulated in cancer tissues in all cases. The up-regulation of DD3 and the down-regulation of IGFBP-3 gene expression in cancer tissue were conserved after RNA amplification. A cDNA microarray also revealed an IGFBP-3 down-regulation in cancer tissue as well as several genes known to be differerentially expressed in prostate cancer. Taken together, we present a novel method for the isolation of intact RNA from laser microdissection-derived prostate cancer tissue useful for downstream applications of real-time RT-PCR and cDNA microarrays
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