NF1 mutation analysis using a combined heteroduplex/SSCP approach

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized predominantly by neurofibromas, café-au-lait spots, and Lisch nodules. The disease is caused by disruptive mutations of the large NF1 gene, with half of cases caused by new mutation. Less than 100 constitutional mutations have thus far been published, ranging from very large deletions to point mutations. We have pursued NF1 mutation analysis by heteroduplex analysis (HDA) and single-strand conformational polymorphism analysis (SSCP) of individual exons. We streamlined these techniques to eliminate the use of radioactivity, to apply both methods to the same PCR product, and to multiplex samples in gels. Applied simultaneously to a set of 67 unrelated NF1 patients, HDA and SSCP have thus far identified 26 mutations and/or variants in 45 of the 59 exons tested. Disease-causing mutations were found in 19% (13/67) of cases studied. Both techniques detected a variety of mutations including splice mutations, insertions, deletions, and point changes, with some overlap in the ability of each method to detect variants.     

Evidence for role of transforming growth factor-beta in RRR-alpha-tocopheryl succinate-induced apoptosis of human MDA-MB-435 breast cancer cells

MDA-MB-435 human breast cancer cells treated with 10 micrograms/ml of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for one, two, three, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respectively. Likewise, cells cultured for one, two, and three days with conditioned media (CM) obtained from MDA-MB-435 cells treated with VES exhibit 10%, 36%, and 74% apoptosis, respectively. A quantitative luciferase-based assay showed CM from VES-treated cells collected at 24 and 48 hours after treatment initiation to contain 75 and 32 pg of active transforming growth factor-beta (TGF-beta), respectively, per 10(6) cells. Although purified TGF-beta 1 is not an effective apoptotic agent for MDA-MD-435 cells, cotreatment of the cells for three days with suboptimal levels of VES (2.5 and 5 micrograms/ml) + 10 ng/ml of purified TGF-beta 1 enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF-beta-signaling pathway by transient transfection of MDA-MB-435 cells with antisense oligomers to TGF-beta type II receptor (TGF-beta R-II) blocked VES-induced apoptosis. Likewise, addition of neutralizing antibodies to TGF-beta 1 or to all three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) blocked VES- and CM-induced apoptosis. Furthermore, inhibitors of TGF-beta conversion from an inactive latent form to a biologically active form inhibited VES-induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF-beta or the TGF-beta receptor-signaling pathway with antisense oligomers or ligand-neutralizing antibodies or prevention of activation of TGF-beta indicates a role for TGF-beta signaling in VES-induced apoptosis.     

Validation of a Caco-2 cell monolayer culture for drug transport studies

The advancement of nursing knowledge to improve clinical plastic surgical nursing practice was identified as a priority for the American Society of Plastic and Reconstructive Surgical Nurses (ASPRSN). The purpose of this investigation was to determine the specific research priorities and perspectives of practicing plastic surgical nurses. In addition, nurses were asked to rank their current concerns for the future of plastic surgical nursing.     

Refined microscopic urinalysis for red blood cell morphology in the evaluation of asymptomatic microscopic hematuria in a pediatric population

PURPOSE: The use of refined microscopic urinalysis for the presence of dysmorphic red blood cells (RBCs) has been evaluated in children and adults with a known source of hematuria. We examined the clinical usefulness of this study in a pediatric population with an unknown source of hematuria. MATERIALS AND METHODS: Children 12 years old or younger referred for evaluation of asymptomatic microscopic hematuria exhibiting 4 or more RBCs per high power field were enrolled in this study. Patients provided a first morning urine sample subjected to refined urinalysis for RBC morphology. Standard evaluation of patients was performed until a final diagnosis of the hematuria source was identified. RESULTS: A total of 44 patients completed the study. Refined urinalysis revealed pure dysmorphic RBCs in 22 patients, pure isomorphic RBCs in 8 and mixed isomorphic/dysmorphic RBCs in 14. The presence of dysmorphic RBCs correctly predicted a glomerulotubular source of hematuria in 29 of 36 patients (sensitivity 83%, specificity 81%), while the presence of isomorphic RBCs predicted a uroepithelial source of hematuria in 2 of 8 patients (sensitivity 25%, specificity 22%). Hematuria and 2+ proteinuria (100 mg./dl.) were more sensitive (100%) and specific (83%) than the presence of dysmorphic RBCs in predicting glomerulotubular hematuria. CONCLUSIONS: We believe that this is a costly test offering little additional information to the evaluation of microscopic hematuria in children. A thoughtful history and physical examination with microscopic urinalysis and dipstick for proteinuria provide an equal amount of diagnostic information. We do not recommend its routine use in the evaluation of microscopic hematuria in children.