RNF4 Is a Coactivator for Nuclear Factor Y on GTP Cyclohydrolase I Proximal Promoter

GTP cyclohydrolase I (GCH) is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH4) that is essential for the synthesis of nitric oxide and catecholamines including dopamine and serotonin. Therefore, the regulation of GCH expression is important in determining the catecholamine levels in the brain under pathophysiological conditions. During the study of human disease dopa-responsive dystonia, we found that coactivator RNF4 is involved in the GCH gene expression. Through serial deletion and mutagenesis studies of the GCH promoter, we defined the RNF4-responsive element on GCH proximal promoter as a CCAAT box. RNF4 did not possess specific DNA binding activity toward this CCAAT box, which suggests that RNF4 may be a coactivator of the CCAAT box-binding protein nuclear factor Y (NF-Y). Cotransfection of a dominant-negative mutant of NF-Y resulted in a significant reduction in RNF4-mediated CCAAT box activation. In addition, overexpression of RNF4 could not activate the CCAAT box in Drosophila melanogaster SL2 cells, which are devoid of endogenous NF-Y, whereas overexpression of RNF4 and NF-Y could. Furthermore, immunoprecipitation experiments revealed the physical association between RNF4 and the NF-Y complex. These data indicate that RNF4 imposes functional importance on GCH promoter

Screening Assay of Very Long Chain Fatty Acids in Human Plasma with Multiwalled Carbon Nanotube-Based Surface-Assisted Laser Desorption/Ionization Mass Spectrometry

Peroxisomal disorders are characterized biochemically by elevated levels of very long chain fatty acids (VLCFAs) in serum. Herein, we describe a novel approach for quantification of VLCFAs in serum, namely, eicosanoic acid (C20:0), docosanoic acid (C22:0), tetracosanoic acid (C24:0), and hexacosanoic acid (C26:0). The methodology is based on (i) enrichment of VLCFA derivatives using multiwalled carbon nanotubes (MWCNTs); (ii) quantification using stable isotope-labeled internal standards; and (iii) direct detection using MWCNT-based surface-assisted laser desorption/ionization-time-of-flight mass spectrometry (SALDI-TOFMS). Four kinds of MWCNTs (Aldrich 636843, 636495, 636509, and 636819) of different lengths and diameters were tested using the developed technique. The data show that 636843, the MWCNT with the largest outer diameter (o.d.), the widest wall thickness, and shortest length, had the best limit of detection (0.5-1 mu g/mL) We also found that there was no significant difference in enrichment efficiency of VLCFAs between the four MWCNTs, which suggests that the size of the MWCNT may contribute to desorption/ionization efficiency. To our knowledge, this is the first study to test the enrichment of VLCFAs using MWCNTs of different sizes. We have shown that the VLCFAs adsorbed by MWCNTs can be analyzed by SALDI-TOFMS. In addition, this method does not require liquid/gas chromatography separation, thereby allowing for high-throughput screening of VLCFAs in peroxisomal disorders

Edge-enhanced radiology with broadband synchrotron X-rays

10.1016/S0168-583X(02)01537-9Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms199436-440NIMB

산화물 음극 표면의 과잉 바륨 형성 메커니즘 규명

22kc

Complex Rearrangements Between Chromosomes 6, 10, and 11 With Multiple Deletions at Breakpoints

Here we report on a girl with minor facial anomalies, cleft palate, seizures, microcephaly, psychomotor retardation, and a congenital heart defect. Complex of cytogenetic methods [GTG-banding, spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), multicolor banding (mBAND), and comparative genomic hybridization (array CGH)] showed complex chromosomal rearrangements (CCRs) involving chromosomes 6, 10, and 11 and 4 deletions at the breakpoints. Her father had an unrelated translocation between chromosomes 3 and 16, suggesting the possibility of an autosomal dominant trait that predisposes to complex synapses and recombination between multiple chromosomes during meiosis. This study demonstrates the power of combining available chromosome analysis technologies in resolving CCR. (C) 2010 Wiley-Liss, Inc

Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies

Experimental treatment of bilateral fetal chylothorax using in-utero pleurodesis

Objective To assess the use and efficacy of in-utero pleurodesis for experimental treatment of bilateral fetal chylothorax. Methods This was a study of 78 fetuses with bilateral pleural effusion referred to three tertiary referral centers in Taiwan between 2005 and 2009. Fetuses were karyotyped following amniocentesis and the lymphocyte ratio in the pleural effusion was determined following thoracocentesis. Forty-nine (62.8%) fetuses had a normal karyotype and were recognized to have fetal chylothorax; of these, 45 underwent intrapleural injection of 0.1KE OK-432 per side per treatment. We evaluated clinical (hydrops vs. no hydrops) and genetic (mutations in the reported lymphedema-associated loci: VEGFR3, PTPN11, FOXC2, ITGA9) parameters, as well as treatment outcome. Long-term survival was defined as survival to 1 year of age. Results The overall long-term survival rate (LSR) was 35.6% (16/45); the LSR for non-hydropic fetuses was 66.7% (12/18) and for hydropic fetuses it was 14.8% (4/27). If we included only fetuses with onset of the condition in the second trimester, excluding those with onset in the third trimester, the LSR decreased to 29.4% (10/34). Notably, 29.6% (8/27) of hydropic fetuses had mutations in three of the four loci examined. Conclusions OK-432 pleurodesis appeared to be an experimental alternative to the gold-standard technique of thoracoamniotic shunting in non-hydropic fetal chylothorax. In hydropic fetuses, pleurodesis appeared less effective. Copyright (C) 2011 ISUOG. Published by John Wiley & Sons, Ltd

Preimplantation and prenatal genetic diagnosis of aromatic L-amino acid decarboxylase deficiency with an amplification refractory mutation system-quantitative polymerase chain reaction

Objectives: To develop a diagnostic platform for preimplantation genetic diagnosis (PGD) and prenatal genetic diagnosis (PND) to prevent births of aromatic L-amino acid decarboxylase deficiency (AADC) patients. Materials and Methods: Five Taiwanese families carrying AADC were enrolled. A novel technique, amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR), was developed for both of PGD and PND. For POD, blastomere biopsies of day-3 cleavage-stage embryos were subjected to ARMS-qPCR. Villi, cultured amniocytes, or both were used to confirm the POD result; this approach could also be used as the sole method for PND after in vivo conception). Results: Unaffected live births were achieved in four of the five families, except one with ongoing POD. The ARMS-qPCR correctly classified blastomeres (from day-3 cleavage-stage embryos) as affected (homozygous mutant), carrier (heterozygous for mutant and wild-type alleles), or normal (homozygous wild-type) within 1 working day. Conclusions: To our knowledge, this is the first report of successful POD of AADC. The molecular technique we devised (ARMS-qPCR) was applicable for POD as well as PND of AADC. Furthermore, it has great potential for similar applications in other monogenic disorders. Copyright (C) 2011, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved