Cobaltous ion inhibition of lipid peroxidation in biological membranes.

The effect of cobalt on lipid peroxidation in biological membranes, phospholipid liposomes and fatty acid micelles was investigated. Cobaltous ion, at micromolar concentrations, inhibited iron-ascorbate induced lipid peroxidation in erythrocyte ghosts, microsomes and phosphatidylserine liposomes at pH 7.4. The pH seemed to be important for the anti-peroxidative effect of cobalt, because under slightly acidic conditions cobalt did not inhibit peroxidation. Cobalt was less effective in inhibiting peroxidation stimulated by organic hydroperoxides. Iron-ascorbate induced lipid peroxidation was also inhibited by EDTA. However, certain ratios of EDTA: cobalt in the reaction mixture stimulated peroxidation. Cobalt did not inhibit lipid peroxidation in linoleic acid micelles and phosphatidylethanolamine liposomes. The presence of phosphatidylserine, however, rendered these micelles and liposomes to cobalt inhibition. We conclude that the cobaltous ion is a potent inhibitor of lipid peroxidation in biological membranes and that the binding of cobalt to phosphatidylserine is necessary for the inhibitory effect of this metal ion.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Finding cancer in primary care outpatients with low back pain: a comparison of diagnostic strategies.

OBJECTIVE: To compare strategies for diagnosing cancer in primary care patients with low back pain. Strategies differed in their use of clinical findings, erythrocyte sedimentation rate (ESR), and plain x-rays prior to imaging and biopsy. DESIGN: Decision analysis and cost effectiveness analysis with sensitivity analyses. Strategies were compared in terms of sensitivity, specificity, and diagnostic cost effectiveness ratios. SETTING: Hypothetical MEASUREMENTS: Estimates of disease prevalence and test characteristics were taken from the literature. Costs were represented by the Medicare reimbursement for the tests and procedures employed. MAIN RESULTS: In the baseline analysis, using magnetic resonance imaging (MRI) as the imaging procedure prior to a single biopsy, strategies ranged in sensitivity from 0.40 to 0.73, with corresponding diagnostic costs of $14 to $241 per patient and average cost effectiveness ratios of $5,283 to $49,814 per case of cancer found. Incremental cost effectiveness ratios varied from $8,397 to $624,781; 5 strategies were dominant in the baseline analysis. Use of a higher ESR cutoff point (50 mm/hr) improved specificity and cost effectiveness for certain strategies. Imaging with MRI, or bone scan followed in series by MRI, resulted in a fewer unnecessary biopsies than imaging with bone scan alone. Cancer prevalence was an important determinant of cost effectiveness. CONCLUSIONS: We recommend a strategy of imaging patients who have a clinical finding (history of cancer, age > or = 50 years, weight loss, or failure to improve with conservative therapy) in combination with either an elevated ESR (> 50 mm/hr) or a positive x-ray, or using the same approach but imaging directly those patients with a history of cancer.

FDDI information management system for centralizing interactive, computerized multimedia clinical experiences in pediatric rheumatology/Immunology.

This paper describes the design, authoring, and development of interactive, computerized, multimedia clinical simulations in pediatric rheumatology/immunology and related musculoskeletal diseases, the development and implementation of a high speed information management system for their centralized storage and distribution, and analytical methods for evaluating the total system's educational impact on medical students and pediatric residents. An FDDI fiber optic network with client/server/host architecture is the core. The server houses digitized audio, still-image video clips and text files. A host station houses the DB2/2 database containing case-associated labels and information. Cases can be accessed from any workstation via a customized interface in AVA/2 written specifically for this application. OS/2 Presentation Manager controls, written in C, are incorporated into the interface. This interface allows SQL searches and retrievals of cases and case materials. In addition to providing user-directed clinical experiences, this centralized information management system provides designated faculty with the ability to add audio notes and visual pointers to image files. Users may browse through case materials, mark selected ones and download them for utilization in lectures or for editing and converting into 35mm slides.