Flow cytometric reporter assays provide robust functional analysis of signaling complexes.

Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB-responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways.

Prevalence and clinical significance of antineutrophil cytoplasmic antibody in Graves' patients treated with propylthiouracil.

Development of antineutrophil cytoplasmic antibody (ANCA) during therapy with propylthiouracil (PTU) is not uncommon and PTU-induced ANCA-positive vasculitis is also reported. The aim of this study was to assess the presence and clinical significance of ANCA positivity in Graves' patients treated with PTU. Newly diagnosed Graves' disease patients (prospective group, n = 58) were evaluated before and during therapy with PTU to investigate the development of ANCA positivity. ANCA positivity is also investigated in previously diagnosed Graves' patients who had already been receiving PTU treatment (cross-sectional group, n = 51). Comparisons with Hashimoto thyroiditis (n = 55) and toxic nodular goitre (n = 20) patients, and healthy control subjects (n = 20) were carried out to define the possible influence of hyperthyroidism and/or thyroid autoimmunity on ANCA positivity. At baseline evaluation, ANCA was negative in all newly diagnosed Graves' patients. Only 28 of the 58 patients in prospective group completed 2 years of follow-up which occurred at 3-month intervals. ANCA positivity was detected 32.1% (n = 9) in a mean period of 11.7 +/- 6.1 months in prospective group. Only two (3.9%) patients in a cross-sectional group had ANCA positivity in a mean treatment period of 7.6 +/- 4.6 months. None of the patients with ANCA positivity developed symptoms and signs related to vasculitis. None of the patients with Hashimoto thyroiditis and toxic nodular goitre, and healthy control subjects had ANCA positivity. PTU therapy is associated with asymptomatic production of ANCA in a time-dependent manner, which mostly disappears after discontinuation of therapy. Hyperthyroidism or autoimmunity per se does not appear to have effect on development of ANCA positivity.

Thymidylate synthase genotype and serum concentrations of homocysteine and folate in Behçet's disease.

The aim of this study was to assess whether thymidylate synthase (TYMS) genotype, serum homocysteine, and folate concentrations were related to venous thrombosis in Behçet's disease (BD) patients. The study included 104 BD patients fulfilling the International Study Group Criteria for the diagnosis of BD and 121 healthy individuals-controls. Out of 104 patients, 50 (48%) had vascular involvement: 34 had active-history of venous thrombosis, 16 had arterial involvement (aneurysm), and 11 of these patients had both venous and arterial lesions as confirmed by Doppler ultrasound and/or angiography. Genotype analysis of the TYMS promoter enhancer region was determined by polymerase chain reaction. The distribution of the TYMS genotypes 2R/2R, 2R/3R, 3R/3R, 4R/2R, and 3R/3R were not significantly different between BD patients and control group (p>0.05; 16.5% vs 8.3%, 49.0% vs 53.9%, 31.7% vs 38.0%, 1.9% vs 0%, and 1.0% vs 0%, respectively). TYMS genotypes were not associated with thrombosis and serum homocysteine concentration in BD patients. The mean serum homocysteine level in patients with thrombosis (14.87+/-8.99 micromol/L) was significantly higher than the level in patients without thrombosis (10.78+/-3.81 micromol/L; p<0.05). Serum folate concentrations were not different between the BD patients and the healthy controls. The study results suggest that the distribution TYMS genotype in BD was not different from that of healthy controls. There was no relationship between TYMS genotype and the homocysteine levels in BD patients with thrombosis or without thrombosis.

The effects of exogenous neuropeptide Y on feeding and sexual behavior in the red-sided garter snake (Thamnophis sirtalis parietalis).

Actively courting adult male red-sided garter snakes were injected with one of two dosages of neuropeptide Y (NPY) or saline into the third cerebral ventricle. Courtship and feeding responses were assessed prior to surgery, 4-5 h and 24 h post-surgery. Feeding behavior was tested by the presentation of a favored food, an earthworm. NPY significantly reduced courtship behavior scores at both low (0.16 nmol) and high (0.77 nmol) doses 4-5 h after surgery. Snakes also took much longer to reach the criterion courtship score at the high dose. Feeding occurred only in snakes treated with NPY. At the low dose 29% ate at 4-5 h and 24 h after surgery; 43% of the snakes treated with the high dose ate 4-5 h post-surgery. When tested the day following injection, animals were courting at levels comparable to presurgery levels and refused food.

Maintenance following a very-low-calorie diet.

The authors posed 2 questions in this randomized study of maintenance procedures in which participants were followed for 15 months after completion of a very-low-calorie diet: Would stimulus narrowing during the reintroduction of solid food, achieved by the use of prepackaged foods, improve weight losses and the maintenance of those losses as compared with the use of regular food? Would reintroduction of foods dependent on progress in losing or maintaining weight be superior to reintroduction on a time-dependent basis? Neither the stimulus narrowing condition nor the reintroduction procedure enhanced either maximum weight loss or maintenance of those losses. The stimulus narrowing condition appeared to be poorly tolerated; compliance and attendance were poorer in this condition than in the regular food condition.