An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function.

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.

Preoperative diagnosis and localization of aldosterone-producing adenoma by adrenal venous sampling after administration of metoclopramide.

BACKGROUND AND PURPOSE: Adrenal venous sampling is the most reliable test to distinguish aldosterone-producing adenoma (APA) from idiopathic hyperaldosteronism (IHA). The diagnostic accuracy can be improved by administration of adrenocorticotropin to minimize pulsatile secretion of aldosterone. Metoclopramide (MCP), a dopamine antagonist, can increase aldosterone secretion promptly without affecting cortisol secretion. This study investigated the diagnostic accuracy of adrenal venous sampling after MCP injection for the preoperative diagnosis and localization of APA. METHODS: Prospective diagnosis and adrenalectomy was based on adrenal venous sampling in 23 patients with a diagnosis of primary aldosteronism. Plasma aldosterone concentrations from adrenal veins and the inferior vena cava were measured before and 30 minutes after intravenous administration of 10 mg MCP. The ratio of bilateral adrenal venous aldosterone concentrations after MCP was used for diagnosis as follows: a ratio greater than 5 indicated APA, less than 3 indicated IHA, and 3-5 indicated an intermediate diagnosis. RESULTS: Catheterization of the right adrenal vein was unsuccessful in three patients. Twelve of 13 patients with an aldosterone ratio greater than 5 after MCP underwent unilateral adrenalectomy, and APA was confirmed in 11 of these patients. One patient with an intermediate diagnosis also had surgically confirmed APA. Six patients had a ratio less than 3. Before MCP administration, 10 of 13 patients with APA had a ratio greater than 5, and three patients had a ratio between 3 and 5; one patient with IHA had a ratio greater than 5. MCP improved the diagnosis of APA to an accuracy of 92% (12/13). Correct diagnosis of APA based on computerized tomography (CT) was 85% (11/13). There was discordance between the findings of adrenal venous sampling and CT in four of 20 patients. CONCLUSIONS: Administration of MCP to stimulate aldosterone secretion during adrenal venous sampling can improve the accuracy of differential diagnosis between APA and IHA.

The detection of the HLA-B27 antigen by immunomagnetic separation and enzyme-linked immunosorbent assay-comparison with a flow cytometric procedure.

The HLA-B27 antigen is an important genetic marker in ankylosing spondylitis (AS). Methods for the detection of B27 include the microlymphocytotoxicity test and, more recently, flowcytometry (FC). Here, we describe a new method, IMS-ELISA, for measuring the B27-antigen. It combines immunomagnetic separation (IMS), to obtain B27-positive cells from whole blood samples, with an enzyme-linked immunosorbent assay (ELISA) as a read-out. IMS-ELISA was tested on 367 samples obtained from five different hospitals in Taiwan. The sensitivity, specificity and accuracy of the method were compared with FC. Any conflicting data between IMS-ELISA and FC was confirmed by HLA-DNA typing via PCR-SSP (polymerase chain reaction-sequence specific primers). Overall, the results for sensitivity, specificity and accuracy obtained by IMS-ELISA and FC did not show any significant difference (p>0.05). However, when considering laboratory time, cost, ease of operation and the screening of large samples for HLA-B27, the IMS-ELISA was superior to the FC method. We conclude that IMS-ELISA may be used as a fast screening method for HLA B27 detection.

Interleukin 8 modulates interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha release from normal human mononuclear cells.

Recombinant human interleukin 8 (IL-8) enhanced the release of inflammatory cytokines including interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) from normal human mononuclear cells in a dose-related manner (from 1 ng/ml to 10 ng/ml with a maximal effect at 5 ng/ml) when the cells incubated with IL-8 for 24 h. This cytokine-releasing activity of IL-8 is temperature-dependent and required protein synthesis since low temperature (4 degrees C) and cycloheximide (100 micrograms/ml) minimized the cytokine release from MNC. However, when IL-8 concentration was greater than 20 ng/ml, the cytokine release was suppressed. For further investigating the subcellular mechanism of the adverse effect of high dose IL-8 (20 ng/ml) in cytokine synthesis, human mononuclear cells (1 x 10(6)/ml) were stimulated with PHA (1 microgram/ml) in the presence of 20 ng/ml IL-8 for 3 days. We found not only [3H]thymidine incorporation of MNC was tremendously inhibited but DNA fragmentation appeared. Subsequently, the cell cycle of PHA-stimulated MNC retarded in the phase of G0/G1. These results suggest that in low concentration (5-10 ng/ml) IL-8 not only activated neutrophil phagocytosis but facilitated the release of inflammatory cytokines from mononuclear cells. Higher dose of IL-8 (more than 20 ng/ml) conversely suppressed these cytokine release from damaged cells by its cytotoxic effect. This newly found cytokine-releasing activity of IL-8 may play a role in the modulation of inflammation.

Prostaglandin E2 suppresses phytohemagglutinin-induced immune responses of normal human mononuclear cells by decreasing intracellular glutathione generation, but not due to increased DNA strand breaks or apoptosis.

Prostaglandin E2 (PGE2) at concentrations more than 1 x 10(-8) M markedly suppressed the cell proliferation and release of soluble molecules of interleukin-2 receptor (sIL-2R), CD4 (sCD4) and CD8 (sCD8) from phytohemagglutinin (PHA)-stimulated normal human mononuclear cells (MNC) in a dose-related manner. To further elucidate the subcellular mechanism of the inhibitory effect of PGE2 on PHA-stimulated MNC, intracellular concentration of glutathione (GSH) in PHA-stimulated MNC was sequentially measured from day 1 to day 3 by enzymic method. Furthermore, the effect of PGE2 on nuclear DNA including DNA strand breaks in alkali treatment and DNA fragmentation (apoptosis) of PHA-stimulated MNC were also measured. We found intracellular GSH levels were significantly decreased in the early stage of lymphocyte activation (day 1), but no evidence of increased DNA strand breaks or apoptotic process appeared in 3-day culture. In addition, butathione sulfoximine (a specific GSH inhibitor) and dibutyryl cyclic AMP also exhibited both proliferation inhibition and GSH-decreasing effects on PHA-stimulated MNC as well as PGE2. These results suggest that the immunosuppressive effect of PGE2 is mediated by the decreased generation of intracellular GSH, but not by the increased DNA strand breaks or apoptotic mechanism in the cells.

Defective expression of neutrophil C3b receptors and impaired lymphocyte Na(+)-K(+)-ATPase activity in patients with systemic lupus erythematosus.

In our previous report, we demonstrated that the functions of phagocytes and lymphocytes were defective in patients with systemic lupus erythematosus (SLE). In an attempt to further clarify the defective mechanisms of these cells, 25 active SLE, 10 bronchial asthma patients (BA) on corticosteroids and 25 age and sex-matched normal individuals were investigated for the expression of membraneous C3b receptors, ionophore-induced 45Ca(2+)-uptake, mitochondrial potentials and phagocytic activity of neutrophils. We found decreased expression of C3b receptors on SLE PMN in both resting (37.2 +/- 3.7% of the normal controls) and FMLP-stimulated (68.3 +/- 7.1% of the normal controls) conditions, whereas the C3b receptor expression on BA-PMN receiving long-term steroid treatment was not different from normal controls. This suggests that the defective phagocytosis of SLE PMN is in the recognition, but not in the ingestion phase because of the normal function of Ca(2+)-influx and mitochondrial activity in SLE PMN. On the other hand, hyporesponsiveness to PHA stimulation (stimulation index: 127.4 +/- 46.3 in SLE vs. 311.2 +/- 30.4 in normals, p = 0.0077) was a distinct cell-mediated immune abnormality in our SLE patients. We measured the membrane potential of individual cells using 3,3'-dihexyloxacarbocyanin and found hyperpolarization in resting SLE lymphocytes. However, the membrane polarization of SLE lymphocytes became lower than that of normal cells after PHA stimulation for 3 days. A similar tendency was also found in Na(+)-K(+)-dependent ATPase activity in SLE lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)