Home About us Contact
|
Home About us Contact | ||
|
|
||
Expression Experiments (expression + experiment)
Selected AbstractsA New Chrna4 Mutation with Low Penetrance in Nocturnal Frontal Lobe EpilepsyEPILEPSIA, Issue 7 2003Tobias Leniger Summary: Purpose: To identify and characterize the mutation(s) causing nocturnal frontal lobe epilepsy in a German extended family. Methods: Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes were screened by direct sequencing. Once a CHRNA4 mutation was identified, its biophysical and pharmacologic properties were characterized by expression experiments in Xenopus oocytes. Results: We report a new CHRNA4 mutation, causing a ,4-T265I amino acid exchange at the extracellular end of the second transmembrane domain (TM). Functional studies of ,4-T265I revealed an increased ACh sensitivity of the mutated receptors. ,4-T265I is associated with an unusual low penetrance of the epilepsy phenotype. Sequencing of the TM1-TM3 parts of the 1 known nAChR subunits did not support a two-locus model involving a second nAChR sequence variation. Conclusions: nAChR mutations found in familial epilepsy are not always associated with an autosomal dominant mode of inheritance. ,4-T265I is the first nAChR allele showing a markedly reduced penetrance consistent with a major gene effect. The low penetrance of the mutation is probably caused by unknown genetic or environmental factors or both. [source] Annexin A1 subcellular expression in laryngeal squamous cell carcinomaHISTOPATHOLOGY, Issue 6 2008V A F Alves Aims:, Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. Methods and results:, Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. Conclusions:, ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases. [source] Regulation of the Murine TRACP Gene PromoterJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2003AI Cassady Abstract The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function. [source] The transcription factor SOX17 is involved in the transcriptional control of the uteroglobin gene in rabbit endometriumJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2007Carlos Garcia Abstract The transcription of the uteroglobin gene (ug) is induced by progesterone in the rabbit endometrium, primarily through the binding of the progesterone receptor to the distal region of the ug promoter. However, other transcription factors participate in the progesterone action. The proximal ug promoter contains several putative consensus sequences for the binding of various progesterone-dependent endometrial nuclear factors (Perez Martinez et al. [1996] Arch Biochem Biophys 333: 12,18), suggesting that several transcription factors might be implicated in the hormonal induction of ug. We report here that one of these progesterone-dependent factors specifically binds to the sequence CACAATG (,183/,177) of the rabbit ug promoter. This sequence (hereafter called element G,) is very similar to the consensus sequence for binding of the SOX family of transcription factors. Mutation of the element G, reduced transcription from the ug promoter in transient expression experiments. The endometrial factor was purified and analyzed by nano-liquid chromatography and ion trap coupled mass spectrometry yielding two partial amino acid sequences corresponding to a region of SOX17 that is highly conserved inter-species. This identification was confirmed by immunological techniques using a specific anti-SOX17 antibody. In agreement with the above findings, overexpression of SOX17 in transfected endometrial cells increased transcription from the ug promoter. SOX17 gradually accumulated in the nucleus in vivo concomitant with the induction of ug expression by progesterone in the endometrium. Thus, these findings implicate, for the first time, SOX17 in the transcriptional control of rabbit ug. J. Cell. Biochem. 102: 665,679, 2007. © 2007 Wiley-Liss, Inc. [source] Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seedsTHE PLANT JOURNAL, Issue 2 2006I. Rubio-Somoza Summary The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5,- GATAAGATA -3,) encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5,-TATCCAC-3,/5,-GTGGATA -3,) encoding a high-pI , -amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans -activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF. [source] Folate synthesis in plants: the last step of the p -aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyaseTHE PLANT JOURNAL, Issue 4 2004Gilles J.C. Basset Summary In plants, the last step in the synthesis of p -aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a , -lyase that converts 4-amino-4-deoxychorismate (ADC) , the reaction product of the PabA and PabB enzymes , to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated. [source] Novel cytochrome P450s, CYP6BB1 and CYP6P10, from the salt marsh mosquito Aedes sollicitans (Walker) (Diptera: Culicidae)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2008Shaoming Huang Abstract Based on the conserved heme-binding region and the charge pair consensus of insect cytochrome P450s, two novel full-length P450 cDNAs, CYP6BB1 and CYP6P10, were cloned from the salt marsh mosquito Aedes sollicitans (Walker). CYP6BB1 and CYP6P10 had open reading frames of 1,518 and 1,521 nucleotides encoding 506 and 507 amino acid residue proteins, respectively. Several alleles with amino acid substitutions were found both in CYP6BB1 and CYP6P10. The deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Sequence analysis showed that both CYP6BB1 and CYP6P10 shared highest sequence identities with P450 CYP6P4, 56% and 65%, respectively. Phylogenetic analysis showed both CYP6BB1 and CYP6P10 were grouped into the clade containing several P450s from subfamily CYP6P. Real-time RT-PCR analysis showed CYP6BB1 but not CYP6P10 transcription in females was significantly increased 24 h after a blood meal. Neither CYP6BB1 nor CYP6P10 were life stage or gender specific. Protein expression experiments are needed to determine the functions of these proteins. Arch. Insect Biochem. Physiol. 2007. © 2007 Wiley-Liss, Inc. [source] Cloning and Sequencing of the Biosynthetic Gene Cluster for Saquayamycin Z and Galtamycin B and the Elucidation of the Assembly of Their Saccharide ChainsCHEMBIOCHEM, Issue 8 2009Annette Erb Abstract Sweet ways: We have investigated the glycosyltransferase genes of the saquayamycin Z (shown) and galtamycin B biosynthetic gene cluster from Micromonospora sp. Tü6368. The results unambiguously show that both compounds are derived from the same cluster. Furthermore, the function of five glycosyltransferases was elucidated, and the results have shed light on the assembly of the sugar chains. The Gram-positive bacterium, Micromonospora sp. Tü6368 produces the angucyclic antibiotic saquayamycin Z and the tetracenequinone galtamycin B. The structural similarity of both compounds suggests a common biosynthetic pathway. The entire biosynthetic gene cluster (saq gene cluster) was cloned and characterized. DNA sequence analysis of a 36.7 kb region revealed the presence of 31 genes that are probably involved in saquayamycin Z and galtamycin B formation. Heterologous expression experiments and targeted gene inactivations were carried out to specifically manipulate the saquayamycin Z and galtamycin B pathways; this demonstrated unambiguously that both compounds are derived from the same cluster. The inactivation of glycosyltransferase genes led to the production of novel saquayamycin and galtamycin derivatives, provided information on the assembly of the sugar chains, and showed that tetracenequinones are formed from angucyclines. [source] | ||||||||||||||||