Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase.

N-dansylhomocysteine (DnsHCys) is quenched on S-nitrosation. The product of this reaction, N-dansyl-S-nitrosohomocysteine, is a sensitive, direct fluorogenic substrate for the denitrosation activity of protein disulfide isomerase (PDI) with an apparent K(M) of 2 microM. S-nitroso-BSA (BSA-NO) competitively inhibited this reaction with an apparent K(I) of 1 microM. The oxidized form of DnsHCys, N,N-didansylhomocystine, rapidly accumulated in cells and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human umbilical endothelial cells and hamster lung fibroblasts were monitored as a function of extracellular BSA-NO concentration via dynamic fluorescence microscopy. The observed quenching of the DnsHCys fluorescence was an indirect measure of cell surface PDI (csPDI) catalyzed denitrosation of extracellular S-nitrosothiols as decrease or increase in the csPDI levels in HT1080 fibrosarcoma cells correlated with the rate of quenching and the PDI inhibitors, 5,5'-dithio-bis-3-nitrobenzoate and 4-(N-(S-glutathionylacetyl) amino)phenylarsenoxide inhibited quenching. The apparent K(M) values for denitrosation of BSA-NO by csPDI ranged from 12 microM to 30 microM. Depletion of membrane N(2)O(3) with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quenching rates of DnsHCys fluorescence by approximately 70%. The K(M) for BSA-NO increased by approximately 3-fold and V(max) decreased by approximately 4-fold. These findings suggest that csPDI catalyzed NO released from extracellular S-nitrosothiols accumulates in the membrane where it reacts with O2 to produce N(2)O(3). Intracellular thiols may then be nitrosated by N2O3 at the membrane-cytosol interface.

Aggregation of Dictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein alpha-subunit, G alpha 2.

The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium's developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase, and phospholipase C. Myristoylation of G protein alpha-subunits is known to affect alpha-subunit association with the beta gamma subunits and membrane localization. The putative site for N-terminal myristoylation of G alpha 2 was mutated from Gly to Ala (G2A) and expressed in the g alpha 2-null cell line, MYC2. Transformants expressing G alpha 2-G2A exhibit physiological and biochemical changes from wild-type cells. G alpha 2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. G alpha 2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. G alpha 2-WT is found in both the pellet and supernatant fractions following lysis of the cells. G alpha 2-G2A however is found almost exclusively in the lysate supernatant. G alpha 2 is radiolabeled upon incubation of cells in [3H]myristate, while G alpha 2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that G alpha 2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated G alpha 2-G2A to the cytosol.

Qualifying for certification in the specialty of occupational medicine.

There are currently a variety of pathways toward certification in occupational medicine. These options may be a source of confusion for potential candidates. As such, this report is offered as a guide for those attempting to complete the requirements for Board eligibility in occupational medicine.

Comparison of four different immunoassays for detection of Toxoplasma-specific immunoglobulin G.

Serum specimens from 246 women of childbearing age were tested for immunoglobulin G (IgG) antibodies to Toxoplasma gondii by four different commercial assays: Abbott IMx microparticle enzyme immunoassay (EIA), Mercia Toxo-G EIA, Bartels Prima EIA, and bioMerieux Vitek (VIDAS) enzyme-linked fluorescent assay. A total of 27 specimens were initially positive with all four assays, 202 specimens were negative, and 17 specimens were discrepant (disagreement among the assays). After repeating tests for the 17 discrepant samples, five resolved (one was positive and four were negative). The 12 remaining discrepant samples were tested by an indirect fluorescent antibody (IFA) assay for Toxoplasma IgG antibodies; five specimens were positive for Toxoplasma IgG by IFA. The resolved sensitivities of the various kits ranged from 88% (bioMerieux VIDAS) to 94% (Abbott IMx), and the specificities were all 98%-99%. These results show that the four serologic tests used for detection of Toxoplasma IgG give very similar results and can all be readily used by clinical laboratories for screening purposes.

Detection of small changes in body composition by dual-energy x-ray absorptiometry.

The ability of dual-energy x-ray absorptiometry (DEXA) to detect small changes in body composition was studied in 17 men and women during a dehydration-rehydration protocol. Scale weight (BW) and total mass (TM) from DEXA were highly related (r > 0.99) as were estimates of fat-free mass (r = 0.99) and percent fat (r = 0.97) from DEXA and densitometry. Changes in BW of approximately 1.5 kg due to fluid loss and gain were highly correlated (r = 0.90) with both changes in TM and soft-tissue mass (STM) by DEXA but less so (r = 0.67) with changes in lean-tissue mass (LTM). Mean changes in TM, STM, and LTM were not different (P > 0.05) from changes in BW. Estimates of bone mass and fat were unaffected by changes in hydration. We conclude that DEXA is able to detect small individual changes in TM and STM and is also useful for detecting group changes in LTM.