We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.
Background/Aims: Recent studies indicate the presence of reactive oxygen species (ROS) producing homologues of the enzymatic subunit (Nox2) of phagocytic NADPH oxidase in non-phagocytic cells. Interestingly, in these cells, ROS produced by the Nox2 homologue(s) was shown to play a role in various regulatory processes, including cell death, proliferation, and aging. The purpose of this study was to investigate whether human cardiomyocytes express Nox2. Methods: The expression of Nox2 was studied in human cardiomyocytes using western blot and immunohistochemical analysis. To analyse the putative expression of Nox2 in human heart disease, cardiac samples from patients who had died subsequent to acute myocardial infarction (AMI) were studied. Results: Both in western blot and immunohistochemical studies, Nox2 expression was found in normal human cardiomyocytes. In patients with AMI, a significant increase in Nox2 expression was found both in viable and in jeopardised cardiomyocytes in the infarcted area. In addition, in the "remote from infarction" area, Nox2 expression was present in cardiomyocytes, but was not increased. Conclusions: Nox2 or its homologue(s) is expressed in normal and jeopardised human cardiomyocytes. This expression is increased in patients with AMI, suggesting a role for this ROS producing Nox2 homologue(s) in the human heart after AMI.
Component Affected g p9 1-phox gp91-phox gp91-phox p22-ph0~ p22-phox p47-ph0~ p67-ph0~ Cytochrome 458 Heme Spectrum
Long interspersed nuclear element-1 (LINE-1) or L1 elements are DNA elements present in the genome in high copy number and capable of active retrotransposition. Here we present a patient with severe chronic granulomatous disease (CGD) caused by insertion of an L1 sequence into intron 5 of the X-lined gene CYBB. Due to internal rearrangements, the insert introduced new splice sites into the intron. This resulted in a highly heterogeneous splicing pattern with introduction of two L1 fragments as new exons into the transcripts and concomitant skipping of exonic coding sequence. Because no wild-type cDNA was found, this mechanism is probably responsible for the patient's phenotype. The L1 fragment, which belongs to the Ta subset of transcriptionally active LINEs, illustrates a new mechanism by which these elements can modify the transcribed coding sequence of genes.
BACKGROUND: Patients with a locally advanced rectal carcinoma benefit from preoperative chemoradiotherapy. MRI is considered the first choice imaging modality after preoperative chemoradiation, although its reliability for restaging is debatable. OBJECTIVE: The purpose of this study was to determine the accuracy of MRI in restaging locally advanced rectal cancer after preoperative chemoradiation. DESIGN: This was a retrospective study. SETTINGS: The study was conducted in a Dutch high-volume rectal cancer center. PATIENTS: A consecutive cohort of 48 patients with locally advanced rectal cancer treated with a curative intent was identified. MAIN OUTCOME MEASURES: Three readers independently evaluated the MRI both for primary staging and for restaging after preoperative chemoradiation and were blinded to results from the other readers as well as histological results. Interobserver variability was determined. Accuracy of the restaging MRI was assessed through the comparison of tumor characteristics on MRI with histopathologic outcomes. RESULTS: T stage was correctly predicted by the 3 readers in 47% to 68% and N stage in 68% to 70%. Overstaging was more common than understaging. Positive predictive values (PPV) among the 3 readers for T0 were 0%, and negative predictive values (NPVs) varied from 84% to 85%. For T1/2, PPVs and NPVs were 50% to 67% and 72% to 90%, and for T3/4 they were 54% to 62% and 33% to 78%. PPVs and NPVs for N0 stage were 81% to 95% and 58% to 73%. Tumor regression grade on MRI did not correspond with histopathologic tumor regression grade; PPVs for good response (tumor regression grade on MRI 1–2) were 48% to 61%, and NPVs were 42% to 58%. Interobserver agreement was fair to moderate for T stage, N stage, and tumor response (κ = 0.20–0.41) and fair to substantial for the relation with the mesorectal fascia (κ = 0.33–0.77). In none of the patients was the surgical plan changed after the restaging MRI. LIMITATIONS: This study was limited by its small sample size and retrospective nature. CONCLUSIONS: MRI has low accuracy for restaging locally advanced rectal cancer after preoperative chemoradiation, and the interobserver variability is significant.
CGD is a rare inherited immunodeficiency syndrome, caused by the phagocytes' inability to produce (sufficient) reactive oxygen metabolites. This dysfunction is due to a defect in the NADPH oxidase, the enzyme responsible for the production of superoxide. It is composed of several subunits, two of which, gp91phox and p22phox, form the membrane-bound cytochrome b558, while its three cytosolic components, p47phox, p67phox and p40phox, have to translocate to the membrane upon activation. This is a tightly and intricately controlled process that involves, among others, several low-molecular weight GTP-binding proteins. Gp91phox is encoded on the X-chromosome and p22phox, p47phox and p67phox on different autosomal chromosomes, and a defect in one of these components leads to CGD. This explains the variable mode of inheritance seen in this syndrome. Clinically CGD manifests itself typically already at a very young age with recurrent and serious infections, most often caused by catalase-positive pathogens. Modern treatment options, including prophylaxis with trimethoprim-sulfamethoxazole and rIFN-gamma as well as early and aggressive anti-infection therapy, have improved the prognosis of this disease dramatically. CGD, as a very well-characterized inherited affection of the hematopoietic stem cells, is predestined to be among the first diseases to profit from the advances in cutting-edge therapeutics, such as gene therapy and in utero stem cell transplantation.
Reactive oxygen species producing NADPH oxidases play important roles under different (patho)physiological conditions. NOX1, NOX2, and NOX4 are important sources of reactive oxygen species in the heart, but knowledge of the calcium-dependent NOX5 in the heart is lacking. The presence of NOX5 was studied via RT-PCR in heart tissue from patients with end-stage heart failure; the tissue was obtained during cardiac transplantation surgery. NOX5 positivity and cellular localization were studied via IHC and digital-imaging microscopy in heart tissues of patients who did not have heart disease and in infarction areas of patients who died of myocardial infarctions of different durations. Furthermore, NOX5 expression was analyzed in vitro by using Western blot analysis. NOX5 RNA was found in the hearts of controls and patients with ischemic cardiomyopathy. In controls, NOX5 localized to the endothelium of a limited number of intramyocardial blood vessels and to a limited number of scattered cardiomyocytes. In infarcted hearts, NOX5 expression increased, especially in infarctions >12 hours, which manifested as an increase in NOX5-positive intramyocardial blood vessels, as well as in endothelium, smooth muscle, and cardiomyocytes. NOX5 was found in cardiomyocyte cytoplasm, plasma membrane, intercalated disks, and cross striations. Western blot analysis confirmed NOX5 expression in isolated human cardiomyocytes. For the first time to our knowledge, we demonstrate NOX5 expression in human intramyocardial blood vessels and cardiomyocytes, with significant increases in the affected myocardium after acute myocardial infarction.
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