Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer

Cytidine-triphosphate synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2.) has been purified over 31 000-fold to homogeneity with 17% recovery from rat liver cytosol, using high-performance liquid chromatography (HPLC) techniques. The presence of CTP synthetase monomer, dimer and tetramer has been demonstrated in the ammonium sulfate fraction of rat liver cytosol. By gel-permeation HPLC, the molecular weights of the three molecular forms of the enzyme have been estimated as 240 000 (tetramer), 120 000 (dimer) and 60 000 (monomer). By gel-permeation chromatography on Bio-Gel A-1.5m column, the molecular weights of dimer and monomer were estimated as 100 000 and 50 000, respectively. The molecular weight of the monomeric subunit is determined to be 66 000 by SDS-polyacrylamide gel electrophoresis. Monomers isolated fresh from 0-30 (NH4)2SO4 fraction of rat liver cytosol are enzymatically active. Purified rat liver CTP synthetase exhibited sigmoidal kinetic plots as a function of the substrate UTP in the presence of the end-product, CTP. Partially purified CTP synthetase usually forms an inactive coagulum on freezing and subsequent thawing. Incubation of CTP synthetase dimer at 25[deg]C for 1 h in the presence of UTP, ATP and Mg2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg2+ are removed by dialysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27539/1/0000583.pd

Analysis of De Novo HOXA 13 Polyalanine Expansions Supports Replication Slippage Without Repair in Their Generation

Polyalanine repeat expansion diseases are hypothesized to result from unequal chromosomal recombination, yet mechanistic studies are lacking. We identified two de novo cases of hand‐foot‐genital syndrome (HFGS) associated with polyalanine expansions in HOXA13 that afforded rare opportunities to investigate the mechanism. The first patient with HFGS was heterozygous for a de novo nine codon polyalanine expansion. Haplotype investigation showed that the expansion arose on the maternally inherited chromosome but not through unequal crossing over between homologs, leaving unequal sister chromatid exchange during mitosis or meiosis or slipped mispairing as possible explanations. The asymptomatic father of the second patient with HFGS was mosaic for a six codon polyalanine expansion. Multiple tissue PCR and clonal analysis of paternal fibroblasts showed only expansion/WT and WT/WT clones, and haplotype data showed that two unaffected offspring inherited the same paternal allele without the expansion, supporting a postzygotic origin. Absence of the contracted allele in the mosaic father does not support sister chromatid exchange in the origin of the expansion. Mosaicism for HOXA13 polyalanine expansions may be associated with a normal phenotype, making examination of parental DNA essential in apparently de novo HFGS cases to predict accurate recurrence risks. We could not find an example in the literature where unequal sister chromatid exchange has been proven for any polyalanine expansion, suggesting that the principal mechanism for polyalanine expansions (and contractions) is slipped mispairing without repair or that the true frequency of unequal sister chromatid exchange involving these repeats is low. © 2013 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97454/1/ajmga35843.pd

Potential association of LMNA-associated generalized lipodystrophy with juvenile dermatomyositis

Abstract Background Juvenile dermatomyositis (JDM) is an auto-immune muscle disease which presents with skin manifestations and muscle weakness. At least 10% of the patients with JDM present with acquired lipodystrophy. Laminopathies are caused by mutations in the lamin genes and cover a wide spectrum of diseases including muscular dystrophies and lipodystrophy. The p.T10I LMNA variant is associated with a phenotype of generalized lipodystrophy that has also been called atypical progeroid syndrome. Case presentation A previously healthy female presented with bilateral proximal lower extremity muscle weakness at age 4. She was diagnosed with JDM based on her clinical presentation, laboratory tests and magnetic resonance imaging (MRI). She had subcutaneous fat loss which started in her extremities and progressed to her whole body. At age 7, she had diabetes, hypertriglyceridemia, low leptin levels and low body fat on dual energy X-ray absorptiometry (DEXA) scan, and was diagnosed with acquired generalized lipodystrophy (AGL). Whole exome sequencing (WES) revealed a heterozygous c.29C > T; p.T10I missense pathogenic variant in LMNA, which encodes lamins A and C. Muscle biopsy confirmed JDM rather than muscular dystrophy, showing perifascicular atrophy and perivascular mononuclear cell infiltration. Immunofluroscence of skin fibroblasts confirmed nuclear atypia and fragmentation. Conclusions This is a unique case with p.T10I LMNA variant displaying concurrent JDM and AGL. This co-occurrence raises the intriguing possibility that LMNA, and possibly p.T10I, may have a pathogenic role in not only the occurrence of generalized lipodystrophy, but also juvenile dermatomyositis. Careful phenotypic characterization of additional patients with laminopathies as well as individuals with JDM is warranted.https://deepblue.lib.umich.edu/bitstream/2027.42/142870/1/40842_2018_Article_58.pd

FÓRUM DE GÁS DO MEDITERRÂNEO ORIENTAL: CONVERGÊNCIA DE PREOCUPAÇÕES DE SEGURANÇA ENERGÉTICA E REGIONAL

The East Mediterranean Gas Forum, a nascent regional organization stands at the crossroads of regional cooperation today. Formed in the wake of natural gas discoveries and the resultant bilateral and multilateral mechanisms, the organization has been beset with positive and negative interdependencies in the region. These interdependencies highlight the energy and regional security concerns of the region’s members. As convergences along energy and regional security lines unfold, the foundational structures of the forum have showcased cracks raising questions regarding its relevance. The study thus seeks to examine the dynamics of the international political economy behind the formation of the EMGF by analysing the convergence of energy and regional security concerns of the specific state actors in the region.O Fórum de Gás do Mediterrâneo Oriental (East Mediterranean Gas Forum — EMGF), uma organização regional nascente, está hoje na encruzilhada da cooperação regional. Formada após as descobertas de gás natural e os mecanismos bilaterais e multilaterais resultantes, a organização tem sido assolada por interdependências positivas e negativas na região. Essas interdependências destacam as preocupações energéticas e de segurança regional dos membros da região. À medida que as convergências sobre segurança energética e regional se desenrolam, as estruturas fundamentais do fórum apresentam rachaduras que levantam questões sobre sua relevância. O estudo procura, portanto, examinar a dinâmica da economia política internacional por trás da formação do EMGF, analisando a convergência das preocupações energéticas e de segurança regional dos atores estatais específicos da região

Novel multilocus imprinting disturbances in a child with expressive language delay and intellectual disability

Multilocus imprinting disturbances (MLID) have been associated with up to 12% of patients with Beckwith-Wiedemann syndrome, Silver-Russell syndrome, and pseudohypoparathyroidism type 1B (PHP1B). Single-gene defects affecting components of the subcortical maternal complex (SCMC) have been reported in cases with multilocus hypomethylation defects. We present a patient with speech and language impairment with mild Angelman syndrome (AS) features who demonstrates maternal hypomethylation at 15q11.2 (SNRPN) as well as 11p15.5 (KCNQ1OT1) imprinted loci, but normal methylation at 6q24.2 (PLAGL1), 7p12.1 (GRB10), 7q32.2 (MEST), 11p15.5 (H19), 14q32.2 (MEG3), 19q13.43 (PEG3), and 20q13.32 (GNAS and GNAS-AS1). The proband also has no copy number nor sequence variants within the AS imprinting center or in UBE3A. Maternal targeted next generation sequencing did not identify any pathogenic variants in ZPF57, NLRP2, NLRP5, NLRP7, KHDC3L, PADI6, TLE6, OOEP, UHRF1 or ZAR1. The presence of very delayed, yet functional speech, behavioral difficulties, EEG abnormalities but without clinical seizures, and normocephaly are consistent with the 15q11.2 hypomethylation defect observed in this patient. To our knowledge, this is the first report of MLID in a patient with mild, likely mosaic, Angelman syndrome.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/173023/1/ajmga62752_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/173023/2/ajmga62752.pd