Protein Cysteinyl-S-Nitrosylation: Analysis and Quantification.

The thiol moiety of cysteine residues can undergo a number of biologic modifications including oxidation, sulfenylation, nitrosylation, persulfidation, metalation, and other modifications. These modifications can control biological function, including gain as well as loss of function. Herein, we focus attention on the proteomic analysis of S-nitrosylation in health and disease. We describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. We present several studies where suitability of this approach for investigating endogenous S-nitrosylation is addressed.

An isolated case of leprosy presenting in a migrant worker in Northern Ireland.

Leprosy was first recorded in 600 bc in India. Europe saw its first cases in the fourteenth century. The worldwide incidence is falling, but the disease can still present in the most unexpected places: this is a report of the first case of leprosy presenting to an emergency department in Northern Ireland. It is important for physicians in both community and hospital medicine to have a high index of suspicion for leprosy in patients with chronic skin conditions who were born outside the UK or other developed countries.

Colchicine and 2-methoxyestradiol Inhibit Human Angiogenesis.

BACKGROUND: Angiogenesis is a critical determinant of tumor growth and the development of metastases. Tubulin inhibitors have been shown to be effective inhibitors of angiogenesis. We hypothesized that colchicine, a well-know tubulin inhibitor and 2-methoxyestradiol (2 MeOH), a novel tubulin inhibitor, would limit the initiation of a human angiogenic response and would limit subsequent neovessel growth in a dose-dependent manner. METHODS: To test this hypothesis, we cultured full-thickness human placental vein discs from three placentas in a fibrin-thrombin clot model. Both colchicine and 2 MeOH were tested over a wide range of concentrations (10(-6) to 10(-12) M) to determine their effect on the percent of wells that initiated an angiogenic response (%I) and the subsequent growth (Angiogenic Index, 0-16 range) of vein-derived neovessels. RESULTS: Colchicine at doses of 10(-6) and 10(-8) M completely inhibited the angiogenic response (CI: 95%, P < 0.0001) but lower (10(-10) to 10(-12) M) doses did not significantly inhibit angiogenesis (P = NS). Effective in vitro colchicine levels far exceed achievable non-toxic human plasma levels. In contrast, 2-methoxyestradiol decreased initiation and angiogenic growth significantly at 10(-6) M (CI: 95%, P < 0.0001), but did not significantly decrease angiogenesis at doses of 10(-8), 10(-10), or 10(-12) M. In contrast to colchicine, human plasma levels of 10(-6) M 2 MeOH are achievable clinically with little or no associated toxicity. CONCLUSIONS: Effective in vitro drug levels of 2 MeOH can be achieved in vivo, suggesting that 2 MeOH may have a role in the clinical treatment of angiogenesis-dependent diseases.

Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants.

Protein disulfide isomerase (PDI) exhibits both an oxido-reductase and an isomerase activity on proteins containing cysteine residues. These activities arise from two active sites, both of which contain pairs of redox active cysteines. We have developed two simple in vivo assays for these activities of PDI, based on the demonstration that PDI can complement both a dsbA mutation and a dsbC mutation when expressed to the periplasm of Escherichia coli. We constructed a variety of mutants in and around the active sites of PDI and analysed them using these complementation assays. Our analysis showed that the active site amino acid residues have a major role in determining the activities exhibited by PDI, particularly the N-terminal cysteine of the N-terminal active site. The roles of the histidine residue at position 38 and the glutamic acid residue at position 30 were also studied using these assays. The results show that these two in vivo assays should be useful for rapid screening of mutants in PDI prior to purification and detailed biochemical analysis.

Mutations in dsbA and dsbB, but not dsbC, lead to an enhanced sensitivity of Escherichia coli to Hg2+ and Cd2+.

The Dsb proteins are involved in disulfide bond formation, reduction and isomerisation in a number of Gram-negative bacteria. Mutations in dsbA or dsbB, but not dsbC, increase the proportion of proteins with free thiols in the periplasm compared to wild-type. We investigated the effects of mutations in these genes on the bacterial resistance to mercuric and cadmium salts. Mutations in genes involved primarily in disulfide formation (dsbA and dsbB) generally enhanced the sensitivity to Hg2+ and Cd2+ while a mutation of the dsbC gene (primarily an isomerase of disulfide bonds) had no effect. Mutations of the dsb genes had no effect on the expression of the mercury-resistance determinants of the transposon Tn501.

Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy.

Thyrotropin releasing hormone (TRH), which stimulates prolactin secretion, increases the fluorescence of cultured bovine anterior pituitary (bAP) cells in the presence of the non-permeant membrane indicator dye FM 1-43 [Stafford SJV. Shorte SL. Schofield JG. (1993) Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep., 13, 9-17]. FM 1-43 is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. The membrane area accessible to FM 1-43 dye, and therefore cell fluorescence, increases during exocytosis as secretory granules fuse with the plasma membrane, and endocytosis as vesicles formed at the plasma-membrane fuse with intracellular organelle membranes. We have here measured changes in FM 1-43 uptake and the intracellular calcium concentration ([Ca2+]i) concurrently in the same cells on exposure to TRH, phorbol myristate acetate (PMA) or NH4Cl. TRH (0.1-10 microM) caused a transient increase in [Ca2+]i in 70-90% of bAP cells and in 60-90% of the responding cells also caused a sustained increased FM 1-43 fluorescence. TRH increased [Ca2+]i but did not affect FM 1-43 fluorescence in GH3 rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D2-receptor agonist quinpirole (10 microM) had little effect on the TRH-induced [Ca2+]i rise in bAP cells, but abolished the increase in FM 1-43 fluorescence. The phorbol ester PMA (0.3-3 microM) caused a small, transient increase in [Ca2+]i followed by a fall to levels lower than original resting levels in 40-60% of bAP cells and increased FM 1-43 uptake in cells showing these changes. Extracellular NH4Cl, which mobilises calcium from an ionomycin-insensitive calcium store, caused a transient [Ca2+]i increase in over 90% of the bAP-cells and increased FM 1-43 uptake in a subpopulation (> 50%) of these. The Na+/H+ ionophore monensin prevented the increase in FM 1-43 fluorescence but not the [Ca2+]i rise induced by TRH, prevented the increases in both FM 1-43 fluorescence and [Ca2+]i caused by NH4Cl, and reduced the number of cells showing a rise in FM 1-43 fluorescence in response to PMA from 64% to 34%. The Ca(2+)-ATPase inhibitor thapsigargin reduced the number of bAP cells displaying TRH-induced increases in [Ca2+]i and membrane-turnover from 74% to 18%, but did not affect the changes in [Ca2+]i or FM 1-43 fluorescence caused by PMA or NH4Cl. We discuss the relationships between the secretogogue-induced increases in FM 1-43 fluorescence and changes in intracellular [Ca2+]i under these conditions.

Thyroliberin-induced changes in the fluorescence of a membrane probe in individual bovine anterior pituitary cells.

1. We have investigated the use of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene) as an indicator of exocytosis in individual bovine anterior pituitary cells using microfluorimetric imaging. 2. TMA-DPH was photolabile in artificial and cell membranes. In cells incubated in TMA-DPH the distribution of fluorescence depended both on the incubation time and the illumination schedule. If the dye was added while the cells were subjected to repeated cycles of 0.36 s light intermittent with 1-15 s dark, the fluorescence of the peripheral annulus and the central region of individual cells rose in parallel and reached a steady state within 200 s; the annulus was always brighter than the central region. However, using long intervening dark periods (200 s), the central region continued to incorporate dye after the annulus had reached a plateau. 3. When the cells were loaded with TMA-DPH using intermittent light with short dark periods, the dye washed out of the central region and the annulus in parallel when external dye was removed. However, if the cells had been loaded using long dark periods, the dye was washed out of the central region more slowly than from the annulus. 4. When cells were incubated in TMA-DPH in the dark for 1 min and then exposed to constant illumination in the presence of external dye, the fluorescence of the central region and the annulus both decayed in parallel to a new steady state. If the cells were incubated in TMA-DPH in the dark for 240 min the fluorescence from each region fell to a steady state but the falls were larger and were not in parallel. 5. We suggest that TMA-DPH fluorescence was derived from plasma membrane-associated and internalized dye and that the amount of fluorescence from the latter varied because TMA-DPH was photobleached. Thus, when illumination was interrupted by short dark intervals, annular fluorescence was high compared to central fluorescence because bleached dye in the plasma membrane was rapidly replaced by unbleached dye from the medium. However, long dark intervals permitted the dye to be internalized before it was bleached and fluorescence was therefore also present in central regions. 6. The total cell fluorescence, observed using 15 s dark intervals, was increased 5-40% (in single cells) in a dose-dependent fashion by addition of TRH (tripeptide thyrotrophin-releasing hormone; 1-200 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells.

The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 microM), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K+. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.

Autoradiographic localization of tritium-labeled dihydrotestosterone in human vas deferens.

The human vas deferens was examined autoradiographically for the presence and distribution of androgen receptors. Samples of vas deferens from the region proximal to the testis and the region at the internal inguinal ring were incubated in vitro with tritium-labeled dihydrotestosterone ([3H]-DHT). Frozen sections of tissue were mounted on autoradiographic emulsion-coated slides and exposed for up to three weeks to demonstrate cells with nuclear accumulations of radioactive hormone. Quantitation of autoradiograms was performed with a Zeiss Videoplan morphometric analysis system. Cells in all five tissue layers of the vas deferens were able to bind androgen receptors in the nucleus, as evidenced by superimposition of silver grains over the nuclei of cells in external, middle, and internal smooth muscle layers, as well as in epithelial and subepithelial stromal cells.

Familial visceral myopathy.

We report on a 13-year-old white body with familial visceral myopathy. The abnormalities of the gastrointestinal and urinary tracts are described and the literature regarding urologic implications of this disorder is reviewed.

Hydronephrosis in the asymptomatic neonate with myelodysplasia.

Early diagnosis and intervention in the child with myelodysplasia can effectively improve and preserve renal function in those newborns presenting with abnormalities at birth or who are at risk for deterioration of renal function from infection, vesicoureteral reflux and/or obstruction. During a 1-year period 10 newborns with myelodysplasia were seen. Hydronephrosis was present in 6, reflux in 3 and urinary tract infection in 3. In each newborn adequate decompression of the bladder and complete resolution of the hydronephrosis were achieved. Uroradiographic evaluation was helpful in determining the best mode of therapy for each individual.

Ultrasonic detection of renal calculi: accuracy tested in an in vitro porcine kidney model.

Porcine kidneys containing human calculi of known size and composition were used to study the ability of ultrasound to localize intrarenal calculi. The study conducted in a blind fashion demonstrated that stones as small as 2 mm could be detected. The chemical composition was not related to the ability to detect the stone. Early trials in the operating room have been encouraging. The porcine kidney model is useful for training purposes.