Release of cell cycle constraints in mouse melanocytes by overexpressed mutant E2F1E132, but not by deletion of p16INK4A or p21WAF1/CIP1

Compared to normal melanocytes, melanoma cell lines exhibit overexpression of hyperphosphorylated retinoblastoma tumor suppressor protein (Rb) or a marked decrease in, or lack of, expression of Rb. Hyperphosphorylation of Rb results in increased E2F-mediated transactivation of target genes and cell cycle progression. Using a combination of gene disruption and ectopic expression in growth factor-dependent mouse melanocytes, we studied the roles of E2F1 and the p16INK4A and p21WAF1/CIP1 CKIs (cyclin dependent kinase inhibitors) in the acquisition of TPA (12-O-tetradecanoyl phorbol-13-acetate)-independent growth in culture, a hallmark of melanomas. Surprisingly, melanocytes from p16INK4A- or p21WAF1/CIP1-null mice remained TPA-dependent, and disruption of p21WAF1/CIP1 accelerated cell death in the absence of this mitogen. Disruption of E2F1 had the most profound effect on melanocyte growth, resulting in a fourfold decrease in growth rate in the presence of TPA. Furthermore, enforced overexpression of the DNA-binding-defective E2F1E132 mutant conferred TPA-independence upon melanocytes and was associated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21WAF1/CIP1. We conclude that neutralization of Rb by E2F1E132, but not the disruption of p16INK4A or p21WAF1/CIP1, resulted in the accumulation of free E2F and cell cycle progression. Thus, mechanisms other than the loss of p16INK4A or p21WAF1/CIP1 that activate E2F may play an important role in melanomas.     

Family functioning and social support in the adaptation of caregivers of children with sickle cell syndromes

OBJECTIVE: To examine moderating effects of family functioning and social support on the relationship of child-related stressors to caregivers' psychological adaptation in a sample of caregivers of children with a chronic illness. METHOD: Participants were 67 caregivers of children and adolescents with sickle cell syndromes. We conducted MANOVAs and subsequent effect size calculations to determine if family functioning would buffer the effects of caring for difficult-to-manage children with this illness. RESULTS: Findings supported a moderator effect of family functioning on the association of children's externalizing behavioral problems to caregivers' symptoms of hostility. Greater levels of cohesive and adaptive family functioning buffered the potential detrimental effects of caring for children perceived as hard to manage. No significant associations were obtained between measures of caregivers' psychological adaptation and the severity of their children's disease. CONCLUSIONS: We make recommendations for family systems interventions, particularly for caregivers of children with behavior problems.     

L-carnitine supplementation in childhood epilepsy: current perspectives

In November 1996, a panel of pediatric neurologists met to update the consensus statement issued in 1989 by a panel of neurologists and metabolic experts on L-carnitine supplementation in childhood epilepsy. The panelists agreed that intravenous L-carnitine supplementation is clearly indicated for valproate (VPA)-induced hepatotoxicity, overdose, and other acute metabolic crises associated with carnitine deficiency. Oral supplementation is clearly indicated for the primary plasmalemmal carnitine transporter defect. The panelists concurred that oral L-carnitine supplementation is strongly suggested for the following groups as well: patients with certain secondary carnitine-deficiency syndromes, symptomatic VPA-associated hyperammonemia, multiple risk factors for VPA hepatotoxicity, or renal-associated syndromes; infants and young children taking VPA; patients with epilepsy using the ketogenic diet who have hypocarnitinemia; patients receiving dialysis; and premature infants who are receiving total parenteral nutrition. The panel recommended an oral L-carnitine dosage of 100 mg/kg/day, up to a maximum of 2 g/day. Intravenous supplementation for medical emergency situations usually exceeds this recommended dosage.     

Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity

Purified mitochondrial cytochrome c oxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear heme a3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a3 nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreased Vmax and a raised Km for oxygen. Under these conditions, the redox state of cytochrome a is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 microM peroxynitrite to 10 microM fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochrome a3, partial reduction of cytochrome a, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 microM) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.     

Improvement of the exercise test after therapy in thyrotoxic periodic paralysis

The diagnosis of periodic paralysis (PP) can be aided by demonstrating a decrease in compound motor action potential (CMAP) amplitude after several minutes of exercise. We report a case of secondary hypokalemic PP due to thyrotoxicosis in which the decrease in the CMAP amplitude after exercise dramatically improved after treatment, when a euthyroid state was achieved. The exercise test is a useful electrophysiologic means of monitoring the neuromuscular status of patients with thyrotoxic periodic paralysis prior to, and after treatment of the thyrotoxicosis.     

Clinicopathologic findings in recurrent choroidal melanoma after transpupillary thermotherapy

OBJECTIVE: This study aimed to report the clinicopathologic findings of recurrent choroidal melanoma after transpupillary thermotherapy (TTT). DESIGN: Two case reports. METHODS: The clinical histories and ophthalmologic findings of two patients with recurrent choroidal melanomas who did not respond to TTT were reviewed. Both patients had their eyes with the melanoma enucleated and processed routinely for light and electron microscopic examination. MAIN OUTCOME MEASURES: The eyes were examined for histopathologic and ultrastructural findings. RESULTS: Histopathologic examination showed choroidal melanomas with extensive tumor necrosis and hemorrhage. In areas, tumor cells appeared histologically intact and presumably viable, with ultrastructural evidence of melanogenesis. The eye from one patient was treated only with TTT and showed hemorrhagic necrosis with cytolysis and no changes to tissues surrounding the melanoma. The eye from the other patient, treated with TTT and a radioactive isotope of iodine (I125) plaque, exhibited areas of intact tumor, tumor necrosis, fibrosis, and radiation retinopathy. CONCLUSIONS: These two cases among a total treated series of 14 patients represent examples of uveal melanoma regrowth after TTT. Opaque media precluded adequate treatment in the first case that showed some TTT effect. The second case showed a combination of TTT and I125 effect and failed because of the aggressive nature of tumor.     

Development of self-handicapping tendencies

This study was undertaken to determine when U.S. children begin to self-handicap, that is, to reduce preparation effort before evaluations rather than applying themselves to do their best. The personal variables examined for their impact on practice behavior were gender, grade level, and self-esteem. The situational variables were time of the self-esteem test (before or after the evaluation task) and importance of the evaluation task. The results showed that (a) the 6th-grade boys were more likely than the 6th-grade girls to self-handicap, (b) the 3rd-grade children were not as affected as the 6th-grade children by the self-evaluation implications of performance evaluations, (c) self-handicapping by low-self-esteem and high-self-esteem 6th graders depended on recent experiences, and (d) the self-affirming experience of a self-esteem test reduced the motivation to self-handicap among high-self-esteem 6th-grade boys.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.