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81.
The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability 总被引:15,自引:0,他引:15
Fanconi anemia (FA) is a rare multi-genic, autosomal and X-linked recessive disorder characterized by hematological abnormalities, developmental defects and increased cancer susceptibility. Patient-derived FA cells display heightened sensitivity to DNA cross-linking agents such as mitomycin C (MMC). In response to DNA damaging agents, and during S-phase of the cell cycle, the FA pathway is activated via the mono-ubiquitination of FANCD2 (FANCD2-Ub), signaling its translocation to discrete nuclear foci, where it co-localizes with the central DNA repair proteins BRCA1 and RAD51. However, the exact function of activated FANCD2-Ub remains unclear. Here, we have characterized the role of the FA pathway in response to DNA replicative stress by aphidicolin (APH) and hydroxyurea (HU). The FA pathway is strongly activated in response to both agents. In addition, using patient-derived FA cell lines and siRNA targeting FANCD2, we demonstrate a functional requirement for the FA pathway in response to low doses of APH: a replicative stress treatment known to result in chromosome breakage at common fragile sites. Both the total number of chromosome gaps and breaks and breaks at the specific common fragile sites FRA3B and FRA16D were significantly elevated in the absence of an intact FA pathway. Furthermore, we demonstrate that APH activates the mono-ubiquitination of both FANCD2 and PCNA and the phosphorylation of RPA2, signaling processive DNA replication arrest. Following APH treatment, FANCD2-Ub co-localizes with PCNA (early) and RPA2 (late) in discrete nuclear foci. Our results demonstrate an integral role for the FA pathway in the DNA replication stress response. 相似文献
82.
83.
Levi MH Bartell J Gandolfo L Smole SC Costa SF Weiss LM Johnson LK Osterhout G Herbst LH 《Journal of clinical microbiology》2003,41(5):2147-2152
The characterization of a novel Mycobacterium sp. isolated from granulomatous skin lesions of moray eels is reported. Analysis of the hsp65 gene, small-subunit rRNA gene, rRNA spacer region, and phenotypic characteristics demonstrate that this organism is distinct from its closest genetic match, Mycobacterium triplex, and it has been named M. montefiorense sp. nov. 相似文献
84.
Christianson SW Greiner DL Deluca D Leif J Phillips NE Hayes SM Hayashi S Joliat MJ Lyons BL Shultz LD 《Journal of autoimmunity》2002,18(2):119-130
Defects in the gene that encodes SHP-1 protein tyrosine phosphatase result in multiple hematopoietic abnormalities and generalized autoimmunity in viable motheaten (me(v)) mice. These mice also exhibit early thymic involution and abnormalities in T cell development. Here, we describe the use of fetal thymic organ culture (FTOC) and bone marrow adoptive transfer to study the effects of SHP-1 deficiency on thymocyte development. Chimeric FTOC established with normal bone marrow placed onto deoxyguanosine-treated fetal thymic lobes or onto scid fetal thymic lobes generated T cells. Bone marrow from SHP-1-deficient me(v)/ me(v) mice generated decreased numbers of T cells in chimeric FTOC established using deoxyguanosine-treated thymi but generated normal numbers in chimeric FTOC established using scid thymi. However, scid fetal thymi seeded with me(v)/ me(v) bone marrow also exhibited morphological abnormalities and contained elevated numbers of macrophages. Addition of IL-7 to me(v)/ me(v) bone marrow-seeded scid FTOC led to increased cell numbers, particularly of macrophages. Intrathymic injection of IL-7 partially restored the ability of progenitor cells in me(v)/ me(v) bone marrow to populate the thymus of adoptive recipients. We conclude that abnormal T cell development in me(v)/ me(v) mice may in part be due to defects in the ability of bone marrow-derived accessory cells to provide bioavailable IL-7 to developing thymocytes. 相似文献
85.
Phylogenetic analysis of the spirochetes Borrelia parkeri and Borrelia turicatae and the potential for tick-borne relapsing fever in Florida 下载免费PDF全文
Schwan TG Raffel SJ Schrumpf ME Policastro PF Rawlings JA Lane RS Breitschwerdt EB Porcella SF 《Journal of clinical microbiology》2005,43(8):3851-3859
Isolates of Borrelia turicatae, Borrelia parkeri, and the Florida canine borrelia (FCB) were examined to further phylogenetically characterize the identities of these spirochetes in the United States. DNA sequences of four chromosomal loci (the 16S rRNA gene, flaB, gyrB, and glpQ) were determined for eight isolates of B. turicatae and six isolates of B. parkeri, which grouped the spirochetes into two distinct but closely related taxa (>98% sequence identity) separate from Borrelia hermsii. The FCB was clearly separated with the group identified as B. turicatae, confirming this bacterium as a relapsing fever spirochete. Therefore, the potential for tick-borne relapsing fever in humans and other animals exists in Florida and future efforts are needed to determine the enzootic hosts and distribution of this spirochete in the southeastern United States. Analysis of plasmids demonstrated both linear and circular forms in B. turicatae but only linear plasmids in B. parkeri, which should be of interest to investigators concerned with plasmid diversity and evolution within this group of spirochetes. 相似文献
86.
Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis 总被引:4,自引:0,他引:4 下载免费PDF全文
Joanne B. Messick Linda M. Berent Sandra K. Cooper 《Journal of clinical microbiology》1998,36(2):462-466
The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats. 相似文献
87.
Seilicovich A Pisera D Sciascia SA Candolfi M Puntel M Xiong W Jaita G Castro MG 《Current gene therapy》2005,5(6):559-572
Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas. 相似文献
88.
The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed. 相似文献
89.
Isolation and RNA-binding analysis of NAD+-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe 总被引:1,自引:0,他引:1
Krebs cycle NAD+-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial
biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated
by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding,
but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiaeCOX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular
in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from
the two yeasts are candidates for involvement in the interaction with RNA.
Received: 19 January 2000 / 24 March 2000 相似文献
90.
Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry 总被引:3,自引:0,他引:3
Yu P Constien R Dear N Katan M Hanke P Bunney TD Kunder S Quintanilla-Martinez L Huffstadt U Schröder A Jones NP Peters T Fuchs H de Angelis MH Nehls M Grosse J Wabnitz P Meyer TP Yasuda K Schiemann M Schneider-Fresenius C Jagla W Russ A Popp A Josephs M Marquardt A Laufs J Schmittwolf C Wagner H Pfeffer K Mudde GC 《Immunity》2005,22(4):451-465
The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype. 相似文献