Studies on singlet oxygen formation and UVA light-mediated photobleaching of the yellow chromophores in human lenses

The protein-bound chromophores, which increase with aging in the human lens, act as UVA sensitizers, producing almost exclusively singlet oxygen in vitro. Direct irradiation of whole, aged human lenses with high intensity UVA light (200 mW cm(-2) for 24 hr), however, failed to produce singlet oxygen damage, as evidenced by the lack of either His or Trp photodestruction. Total homogenates of human lenses prepared in a cuvette under air did show destruction of His and Trp residues by UVA light, but no destruction was seen when equivalent homogenates were prepared under argon. These data are consistent with the idea that the low oxygen levels in the lens prevent singlet oxygen damage in vivo.UVA irradiation of aged human lenses in culture caused an extensive photobleaching of the yellow chromophores. A time course indicated that the photobleaching increased with time, with significant color loss apparent after 6 hr. Homogenization of the irradiated and dark control lenses in 6 M guanidine-HCl, followed by determination of the difference spectrum, showed approximately 50% bleaching of compounds with a lambda(max) at 355 nm. Similarly, fluorophores with a lambda(max) for excitation of 355 nm and for emission of 420 nm were 50% destroyed by the UVA light. Similar results were obtained in vitro by the anaerobic irradiation of a sonication-solubilized WI fraction from type II brunescent cataracts and from aged human lenses. In this system, there was an initial bleaching of 15% after 30 min of irradiation, followed by a slow increase over the next 6 hr to a final bleaching of 30%. The addition of 1.0 m M ascorbic acid, but not 1.0 m M glutathione (GSH), increased the photobleaching to 60% under argon, and the loss of ascorbate could be detected under these anaerobic conditions. In the presence of air, UVA light produced no photobleaching, but rather caused a three-fold increase in absorbance at 345 nm, which was prevented by the inclusion of 1.0 m M ascorbic acid and almost 50% inhibited by 1.0 m M GSH.The data are consistent with the conversion of the triplet state of the sensitizers to anion and cation radicals in the absence of oxygen. Photobleaching may occur either by dismutation of the anion radical or by reduction of the anion radical by ascorbate via type I chemistry. UVA irradiation of an enriched fraction of sensitizers from a proteolytic digest from type II cataract lenses produced a 63% bleaching at 330 nm in the absence of oxygen, and the almost complete loss of the A(330) absorbing and 350/450 nm fluorescent peaks upon HPLC separation. This loss correlated with the loss of the ability of the irradiated fraction to produce singlet oxygen in vitro upon subsequent UVA irradiation.     

Pain perception and effectiveness of the eutectic mixture of local anesthetics in children undergoing venipuncture.

The emulsion of lidocaine and prilocaine (EMLA) is effective in preventing the pain of venipuncture in children. It is therefore important to identify children who could benefit the most from EMLA. We studied the safety and feasibility of two methods of application of EMLA (patch and cream) in a randomized, open-label trial of EMLA patch versus EMLA cream in 160 children with chronic diseases undergoing venipuncture. EMLA patch or cream was applied 60 to 120 min before puncture. Pain was assessed by the children using a visual analogue score. Children also scored the pain of their last puncture and the pain of removing the tape. EMLA patch and cream had similar efficacy (visual analogue scores for the venipuncture were 8.5 +/- 16 and 9.5 +/- 17 out of 100, respectively). Side effects occurred in similar frequencies in the two groups. Adhesiveness of the patch was less effective than that of the cream with Tegaderm. Age was a major determinant of pain perception; younger children recalled more severe pain in their previous puncture. Children recently diagnosed had higher visual analogue scores than those with a long history of chronic disease. We concluded that EMLA patch and cream have a similar efficacy in children undergoing venipuncture. Young children recently diagnosed with chronic disease are most likely to benefit from EMLA.     

Early predictors for lower extremity amputation in a diabetic population: results of a case-controlled study

Lower extremity amputation is devastating to any person and is a very real complication for a diabetic. Identifying early risk factors is tantamount to saving a limb. This study used the national hospital discharge survey to identify 2206 control patients and 2098 diabetic patients with a lower extremity amputation. These patients were not limited based on gender or race as in previous studies. The results show that nephropathy and retinopathy are early predictors.     

Temporal dynamics of the partial pressure of brain tissue oxygen during functional forepaw stimulation in rats

The partial pressure of tissue oxygen (pO2) was measured in rat somatosensory cortex during periodic electrical forepaw stimulation of either 1 min or 4 s in duration, and correlated with simultaneous laser Doppler flowmetry. For both stimulus durations, a transient decrease in tissue pO2 preceded blood flow changes, followed by a peak in blood flow and an overshoot in tissue pO2. With protracted stimulation, tissue pO2 remained only slightly above pre-stimulus baseline, while blood flow was maintained at a reduced plateau phase. A sustained post-stimulus undershoot in tissue pO2 was observed only for the 1 min stimulus. These findings suggest a complex dynamic relationship between oxygen utilization and blood flow.     

Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting

BACKGROUND: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. METHODS: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. RESULTS: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism CONCLUSIONS: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.     

Resistance to enfuvirtide, the first HIV fusion inhibitor

Fusion inhibitors are a new class of antiretroviral drugs (ARVs) for the treatment of human immunodeficiency virus infection. Enfuvirtide is the first in this class to reach market approval. Fusion inhibitors block the last step in the three-step viral entry process consisting of attachment, co-receptor binding and fusion, thereby preventing viral capsid entry into the host cell. Enfuvirtide has a unique mechanism of action and high viral target specificity, and in clinical trials has been shown to exhibit both high efficacy and low toxicity. Enfuvirtide is a peptide mimetic of an essential region within viral envelope glycoprotein gp41 that functions by blocking gp41 structural rearrangements at a transitional pre-fusion conformation. Although different clinical isolates show variation in susceptibility to enfuvirtide, primary resistance has not been observed, and thus enfuvirtide-naive isolates remain clinically sensitive. Acquired resistance centres round a 10 amino acid motif between residues 36 and 45 in gp41 that forms part of the binding site of enfuvirtide. The 10 amino acid motif is critical for viral fusion, and enfuvirtide-resistant mutants show poor replicative capacity compared with wild type. Reversion to a wild-type, drug-sensitive state has been reported following enfuvirtide withdrawal.     

Project ENABLE: a palliative care demonstration project for advanced cancer patients in three settings

At the end of the 1990s, based on data from two major studies of end-of-life (EOL) care, the Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatment (SUPPORT), and the Hospitalized Elderly Longitudinal Project (HELP), a consensus panel report documented the problems and needs of patients with cancer and other life-limiting diagnoses at end-of-life. A national program of The Robert Wood Johnson Foundation (RWJF), Promoting Excellence in End-of-Life Care, attempted to address these needs by funding demonstration projects to test various approaches to improve identified deficits. In 1998, Project ENABLE (Educate, Nurture, Advise Before Life Ends), one of four RWJF-funded cancer center/hospice collaborations of the Promoting Excellence program, began to address these issues. The jointly sponsored Norris Cotton Cancer Center (NCCC)/Hospice of Vermont and New Hampshire (Hospice VNH) program provided an integrated approach to the management of life-limiting cancer. Project ENABLE was aimed at alleviating the symptoms of disease and treatment, enhancing clinician and patient/family communication, offering support for families, friends and other caregivers, addressing emotional and spiritual needs of dying people and providing conceptual and administrative structure to provide EOL care consistent with patients' values and preferences. Although patient symptom data is not yet available, other measures of success included improved access to hospice and palliative care services from the time of diagnosis and a sustained palliative care program at two of the three sites in which the program was implemented.