[EFFECT OF EDUCATIONAL LEAFLETS ON KNOWLEDGE AND ATTITUDE TO TUBERCULOSIS AMONG HOMELESS PERSONS IN TOKYO, JAPAN].

SETTING: Delay in seeking care is one of the critical issues in tuberculosis (TB) control among homeless persons in Japan. Yet knowledge of and attitude towards TB among homeless persons have remained unclear and limited efforts have been made to disseminate information related to TB among homeless persons. OBJECTIVE: To evaluate the effect of TB leaflets, produced and distributed to homeless persons by a group of ex-homeless TB patients, and to understand what homeless persons know about TB. DESIGN: Self-administered questionnaire was conducted among homeless persons before and after distribution of the TB leaflets. Changes in the responses to each question were also subjected to principal component analysis to group questions into types according to response patterns and identify constructs of TB-related knowledge. RESULTS: Results of 88 participants were analyzed. TB knowledge score related to risks and symptoms significantly improved after the intervention (from 54.3% to 70.6%, p < 0.05), while knowledge on treatment cost did not. Two components were identified, namely, the "improvement in TB impression" and "improvement in TB knowledge". CONCLUSION: TB leaflets were effective in improving certain aspects of TB knowledge. However, its effect on knowledge regarding treatment cost, which may be crucial in improving delay, was limited and thus the messages need to be revised.

Coexistence of nodular regenerative hyperplasia of the liver and pulmonary arterial hypertension in patients with connective tissue diseases: report of three cases and review of the literature.

Nodular regenerative hyperplasia of the liver (NRH) is known to be a rare condition in patients with connective tissue diseases (CTD). In this report, we document three patients with CTD who had both NRH and pulmonary hypertension (PH). All three patients developed PH during their course and thereafter developed NRH. The clinical course of these patients suggests that circulatory disturbance caused by PH might be a trigger for NRH.

Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein.

Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.

Novel autoantibodies against 7SL RNA in patients with polymyositis/dermatomyositis.

OBJECTIVE: Autoantibodies against signal recognition particle (SRP) are detected in patients with polymyositis/dermatomyositis (PM/DM). The SRP consists of 7SL RNA and 6 protein components. We examined autoantibodies against deproteinized 7SL RNA in PM/DM patients with anti-SRP antibodies and evaluated the association of anti-7SL RNA antibodies with PM/DM clinically and serologically. METHODS: Sera from 10 Japanese and 22 North American PM/DM patients with anti-SRP antibodies were tested for the presence of anti-7SL RNA antibodies, using the sera to immunoprecipitate deproteinized RNA extracts derived from HeLa cells. RESULTS: The immunoprecipitation analysis indicated that 5 Japanese (50%) and one North American (5%) patient with anti-SRP antibodies had novel autoantibodies against deproteinized 7SL RNA. The frequency of anti-7SL RNA antibodies was significantly higher in Japanese than North American patients (p = 0.006). The presence of anti-7SL RNA antibodies appeared to be associated with DM (2 patients) and finger swelling (2 PM patients). The seasonal onset of the disease was different (p = 0.008) for Japanese PM/DM patients with anti-7SL RNA antibodies, who developed the disease between October and January (mean month November; p = 0.01) from that of patients without these antibodies, who developed it between June and August (mean month July; p = 0.01). CONCLUSION: Novel autoantibodies against 7SL RNA were identified in patients with PM/DM, and the presence of these antibodies was correlated to ethnic background, clinical features, and season of disease onset. These findings indicated that autoantibodies against 7SL RNA are a novel serological marker for a subset of PM/DM cases.

A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk.

When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.

A new method for detecting single nucleotide polymorphism using GFP-display.

The single nucleotide polymorphism (SNP) of aldehyde dehydrogenase-2 (ALDH2) codon 487, GAA (Glu) or AAA (Lys), was examined using green fluorescent protein (GFP)-display, an electrophoretic detection method for single amino acid changes. Although no shift in migration between the GFP-ALDH (Glu487) and GFP-ALDH (Lys487) fusion proteins was observed on SDS/urea gel, the two migrated to different positions when tagged with Asp. The SNP analysis was performed with GFP-ALDH-Asp3, and GFP-ALDH-Asp3 constructed from donors having the codon GAA/GAA, GAA/AAA or AAA/AAA was detected as different patterns as expected. GFP-display is potentially a unique method in SNP analysis, which does not require any special equipment or chemicals.

General properties of GFP-display, an electrophoretic analysis for single amino acid changes in target polypeptides.

The migrating position of green fluorescent protein (GFP)-fused polypeptide varied on an SDS/urea gel by a single amino acid change in the fused polypeptide segment. An easy detection method for a single amino acid change based on this observation was called "GFP-display." Using various target polypeptides, staphylococcal protein A (SpA), Ras, p53, and human beta3 adrenergic receptor (AR), and their mobility-shift patterns resulting from the single amino acid changes, several important properties of GFP-display were revealed as follows: (i). since the binding of dodecyl sulfate ions to acidic or hydrophilic amino acids is weaker than that to basic or hydrophobic amino acids, the ions bound weakly to the fused polypeptide segment are forced to come off by high concentrations of urea prior to the ions bound strongly, resulting in the mobility shift, (ii). the mobility shift is estimated to a certain extent using a new parameter called the "GD value" calculated from the isoelectric point, hydrophilicity, and number of fused amino acids, and (iii). the fluorescence intensity of GFP-fused polypeptide tends to increase with the average hydrophilicity of the fused polypeptide segment. GFP-display will be a helpful technique for many kinds of gene or protein studies related to amino acid substitutions such as the random mutagenesis in a gene of interest.

Production of macrophage inflammatory protein 3alpha (MIP-3alpha) (CCL20) and MIP-3beta (CCL19) by human peripheral blood neutrophils in response to microbial pathogens.

Effects of bacterial pathogens on the production of macrophage inflammatory protein 3alpha (MIP-3alpha) and MIP-3beta from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.

A highly sensitive assay for proteases using staphylococcal protein A fused with enhanced green fluorescent protein.

Enhanced green fluorescent protein (EGFP) was fused with staphylococcal protein A (SpA) and used as a substrate for proteases. An SpA-EGFP assay was done in three steps: (i) digestion of SpA-EGFP by proteases, (ii) addition of rabbit IgG immobilized on Sepharose beads, and (iii) measurement of the fluorescence intensity of supernatant. The assay was sensitive enough to measure picogram levels of trypsin and chymotrypsin, and may be applicable to various other proteases as one of the most sensitive methods.

Application of green fluorescent protein to affinity electrophoresis; affinity of IgG-binding domain C from streptococcal protein G to mouse IgG1.

Affinity electrophoresis (AEP) using green fluorescent protein (GFP) was studied. We constructed a fusion protein that linked S147PGFP and IgG binding domain C from streptococcal protein G (GFP-SpGC). The affinity of GFP-SpGC for mouse IgG1 was measured. The AEP using GFP does not require a staining step after electrophoresis, and was successful with a non-purified sample. Therefore, this method is simple and useful for measuring many samples such as those used in mutational studies.

[Infliximab in the treatment of rheumatoid arthritis].

Infliximab is a chimeric monoclonal antibody capable of neutralizing human TNF alpha. A number of clinical trials for the treatment of rheumatoid arthritis(RA) with infliximab indicated that TNF alpha blockade was effective and well tolerated with the excellent results occurring at 3 and 10 mg/kg in combination with methotrexate. Treatment of RA patients with the combination of infliximab and methotrexate also prevented radiographic evidence of progression of joint damage. If the clinical efficacy is sustained and the safety is confirmed over the long term, infliximab may become an essential agent of choice for the treatment of RA.