Assignment of the gene for methylthioadenosine phosphorylase to human chromosome 9 by mouse-human somatic cell hybridization.

The purine and polyamine metabolic enzyme methylthioadenosine (MeSAdo) phosphorylase is abundant in normal cells and tissues but is lacking from many human and murine malignant cell lines and from cells of some human leukemias in vivo. To explore the genetic control of MeSAdo phosphorylase expression, we measured levels of the enzyme in somatic cell hybrids prepared by fusing MeSAdo phosphorylase-deficient mouse L cell lines with human fibroblasts. In the hybrid clones, MeSAdo phosphorylase activity segregated concordantly with adenylate kinase 1, a marker for human chromosome 9, but not with enzyme markers for any other human chromosome. In hybrid clones derived from human fibroblasts with a reciprocal translocation between chromosomes 9 and 17, MeSAdo phosphorylase activity was confined to cells containing the 9pter----9q12 region. In every case, the enzyme-positive hybrid clones displayed bands of MeSAdo phosphorylase activity with isoelectric points characteristic of both the human and murine enzymes. These results indicate that the structural gene for human MeSAdo phosphorylase, designated MTAP, can be assigned to the 9pter----9q12 region of human chromosome 9. Furthermore, these studies with interspecies somatic cell hybrids show that the MeSAdo phosphorylase-deficient state is recessive in mouse L cell lines.     

Sialidosis and galactosialidosis: chromosomal assignment of two genes associated with neuraminidase-deficiency disorders.

The inherited human disorders sialidosis and galactosialidosis are the result of deficiencies of glycoprotein-specific alpha-neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18; sialidase) activity. Two genes were determined to be necessary for expression of neuraminidase by using human-mouse somatic cell hybrids segregating human chromosomes. A panel of mouse RAG-human hybrid cells demonstrated a single-gene requirement for human neuraminidase and allowed assignment of this gene to the (pter----q23) region of chromosome 10. A second panel of mouse thymidine kinase (TK)-deficient LM/TK- -human hybrid cells demonstrated that human neuraminidase activity required both chromosomes 10 and 20 to be present. Analysis of human neuraminidase expression in interspecific hybrid cells or polykaryocytes formed from fusion of mouse RAG (hypoxanthine/guanine phosphoribosyltransferase deficient) or LM/TK- cell lines with human sialidosis or galactosialidosis fibroblasts indicated that the RAG cell line complemented the galactosialidosis defect, but the LM/TK- cell line did not. This eliminates the requirement for this gene in RAG-human hybrid cells and explains the different chromosome requirements of these two hybrid panels. Fusion of LM/TK- cell hybrids lacking chromosome 10 or 20 (phenotype 10+,20- and 10-,20+) and neuraminidase-deficient fibroblasts confirmed by complementation analysis that the sialidosis disorder results from a mutation on chromosome 10, presumably encoding the neuraminidase structural gene. Galactosialidosis is caused by a mutation in a second gene required for neuraminidase expression located on chromosome 20.     

Diagnostic delay in Horton's disease. Analysis of the diagnostic delay based on a retrospective study of 130 cases]

Temporal arteritis (Horton's disease) is a multifocal granulomatous arteritis which affects elderly people. Its prognosis depends upon the risk of blindness. In a retrospective study of 130 patients we paid special attention to the delay in diagnosis and its relation to the occurrence of ophthalmic complications. In 73 patients (56.2%), this delay exceeded 3 months and reached more than one year in 22 of them. It was not influenced by age or sex. In requests for admission, the diagnosis of temporal arteritis was suggested in only 28 of the 130 cases; 17.7% of the patients were blind. There was a significant increase in the occurrence of ophthalmic lesions when the diagnosis was delayed by 2 to 6 months. In 6 cases, blindness had been preceded by transient amaurosis. These findings confirm that temporal arteritis is belatedly diagnosed by practitioners and that blindness could be avoided by an earlier diagnosis.     

Artificial mobile DNA element constructed from the EcoRI endonuclease gene.

There exist several examples of mobile group I introns. These introns appear to use a straightforward mechanism to achieve highly site-specific and efficient insertion into homologous intronless genes. Because the only intron-specific function required by the prevailing model for the mechanism of intron mobility is the introduction of a site-specific double-stranded break in the intronless recipient DNA molecule, we reasoned that it should in principle be possible to construct artificially mobile DNA sequences. We have constructed an artificial mobile element from the gene for the restriction enzyme EcoRI that is capable of site-specific insertion at rates near those of authentic mobile introns. The generality of the mobility mechanism may enable high-efficiency targeted gene replacements or disruptions in a variety of organisms.     

Mentoring Youth at High Risk: The Perspectives of Professional Mentors

Background

Youth mentoring programs rely largely on volunteers, but youth facing significant risks may be poor candidates for volunteer-based interventions. Full-time “professional” mentors in highly structured programs may be better suited to partner effectively with such youth and their families, but few studies examine professional mentoring interventions. Because of mentoring’s inherent flexibility, mentors’ role conceptualizations can profoundly influence the nature of their work. Serving as a professional mentor may have important implications for how mentors conceptualize and perform their role.

Objective

This qualitative study examined the role conceptions of professional mentors serving at-risk youth.

Methods

Semi-structured interviews with mentors were transcribed, coded, and subjected to thematic analysis.

Results

Mentors described the importance of “professionalism” in prioritizing mentoring, expending considerable effort, and performing difficult or unpleasant tasks. They reported that serving multiple children full-time enabled them to rapidly build expertise, that credibility and authority granted them because of their professional status facilitated their work across multiple key contexts, and that their expertise and long-term commitment facilitated the development of deep relationships. Mentors perceived their role as highly challenging but reported high self-efficacy. They described high multifaceted organizational support, a community for youth, and an individualized child focus.

Conclusions

A mentoring model delivered by experienced professional mentors may hold promise for working with youth at high risk. The role conceptualizations of mentors and the organizational culture within which mentors work may be important in helping youth succeed.     

A comparison of two modalities of stereotactic body radiation therapy for peripheral early-stage non-small cell lung cancer: results of a prospective French study

Objectives:This prospective, observational, non-randomized multicentric study was conducted to compare efficiency and toxicity using different modalities of stereotactic body radiation therapy (SBRT) in early-stage peripheral non-small cell lung cancer (NSCLC).Methods:From 9 April to 11 December, 106 patients were treated according to the local equipment availability for peripheral NSCLC with SBRT: 68 by linear accelerator equipped for SBRT and 38 by Cyberknife^®. Multivariate analysis and propensity score analysis using Inverse Probability Treatment Weighting (IPTW) were undertaken in an effort to adjust for potential bias due to non-randomization.Results:2-year local control rates were 97.0% (95% CI: [90.6%; 99.4%]) with SBRT by Linac vs 100% (95% CI: ([100%; 100%]) with Cyberknife^® (p = 0.2839). 2-year PFS and 2-year OS rates were 52.7% (95% CI [39.9%;64.0%]) versus 54.1% (95% CI [36.8; 68.6%]) (p = 0.8582) and 65.1% (95% CI: [51.9%; 75.5%] versus 83.9% (95% CI: [67.5%; 92.4%] (p = 0.0831) using Linac and Cyberknife^® respectively. Multivariate regression analysis indicates no significant effect of SBRT treatment type on PFS or OS. Local relapse could not be modeled due to the small number of events (n = 2). Acute and late toxicity rates were not significantly different. After IPTW adjustment, results were unchanged.Conclusions:No difference in efficiency or toxicity was shown after SBRT of peripheral NSCLC treatment using Linac or Cyberknife^®.Advances in knowledge:This is the first large prospective non-randomized study focusing on peripheral localized NSCLC comparing SBRT using an appropriately equipped linac with Cyberknife^®. No significant difference in efficiency or toxicity was shown in this situation.     

Genetic variants of ApoE and ApoER2 differentially modulate endothelial function

It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE–ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte–endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2^−/− mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2^−/− mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.Cardiovascular disease risk is modified by common genetic variants of apolipoprotein E (apoE) and its receptor apolipoprotein E receptor 2 (ApoER2), which is a member of the LDL receptor family. Compared with the most common allele apoE3, individuals with the apoE4 allele have an increased risk of atherosclerosis and coronary heart disease (1, 2). The LRP8 gene, which encodes ApoER2, is a major gene locus for premature atherosclerosis and acute myocardial infarction identified in four independent human populations. In particular, homozygous carriers of the ApoER2-R952Q variant have a twofold increased risk of these conditions (3–5). ApoER2-R952Q also has an additive effect with apoE4, with the combined genotype QQ/E4 showing a 3.9-fold greater susceptibility to cardiovascular disease (5). ApoER2 polymorphism-associated risk is independent of cholesterol levels (3–5), and although apoE4 may impact LDL abundance (2), there is also evidence that apoE4-associated risk goes well beyond changes in lipoprotein status (6–9). Whereas there is considerable understanding of the biology of the apoE–ApoER2 tandem in the central nervous system and in Alzheimer’s disease (10), the basis for the cardiovascular impact of the receptor and apoE variants remains unclear.Our prior work demonstrated that ApoER2 is expressed in endothelial cells, where it plays a critical role in the pathogenesis of the antiphospholipid syndrome (APS) (11). The receptor is enriched in caveolae/lipid rafts in which signaling molecules regulating endothelial NOS (eNOS) are compartmentalized (12, 13). We now know that in APS, antiphospholipid antibody recognition of the cell surface protein β2-GPI on endothelial cells promotes β2-GPI dimerization and interaction with the extracellular domain of ApoER2, causing the activation of PP2A and eNOS antagonism. The resulting decrease in bioavailable NO underlies APS-related thrombosis (11). However, the normal function of the receptor in endothelium, and whether and how it modulates apoE actions on endothelium, are unknown.In addition to regulating thrombogenesis, eNOS-derived NO plays a major role in cardiovascular protection via promotion of the integrity of the endothelial cell monolayer and attenuation of endothelial cell–leukocyte adhesion (13). Recognizing that eNOS enzymatic activity is both positively and negatively modulated by signaling molecules in endothelial caveolae/lipid rafts (14), to better understand the biology of ApoER2 in endothelium we hypothesized that apoE3 binding to the receptor activates eNOS. Experiments were performed in cell culture and in mice to test this hypothesis and to determine whether genetic variants in apoE or ApoER2 disrupt this process and thereby adversely impact endothelial function.