Glyceraldehyde 3-phosphate dehydrogenase is a novel autoantigen leading autoimmune responses to proliferating cell nuclear antigen multiprotein complexes in lupus patients

Using 2-dimensional electrophoresis and ion-pair chromatography, we have identified elements of proliferating cell nuclear antigen (PCNA) multiprotein complexes that are reactive to antibodies in sera from patients with systemic lupus erythematosus. Among the various elements of the complexes, a 37 kDa protein (PI 8.5) that specifically reacted with SLE sera, but not with sera from patients with other connective tissue diseases, was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Immunoblot analysis showed that SLE sera reactive with the 37 kDa protein specifically reacted with GAPDH, as did anti-GAPDH mAbs. The purified autoantibodies to GAPDH from lupus serum showed both nuclear speckled and cytoplasmic staining patterns in immunofluorescence on Hep-2 cells. In addition, enzyme-linked immunosorbent assay (ELISA) revealed the presence of anti-GAPDH autoantibodies in 47% of lupus patients. Longitudinal analysis of the reactivity of lupus sera to PCNA complexes showed the autoimmune response to spread from GAPDH to other elements of PCNA complexes, and the presence of anti-GAPDH antibodies was significantly correlated with increased levels of serum PCNA. Taken together, these findings suggest that GAPDH interacting with PCNA in association with its cellular function is a novel autoantigen recognized by lupus sera, and that GAPDH thus plays an important role in the induction of autoimmune responses against the PCNA complex.     

Relationships between the serum levels of soluble leptin receptor and free and bound leptin in non-pregnant women of reproductive age and women undergoing controlled ovarian hyperstimulation

BACKGROUND: The aim of this study was to investigate the relationships between the serum levels of soluble leptin receptor (SLEPR), and total, free and bound leptin, and the change in the serum SLEPR level during an IVF cycle. METHODS: Serum concentrations of leptin and SLEPR were measured in 50 Japanese women of reproductive age, and 20 patients participating in an IVF programme. The total leptin was fractionated into free and bound portions by gel filtration chromatography. RESULTS: The SLEPR level was negatively correlated with the body mass index (BMI) (r = -0.548, P < 0.0001), total leptin (r = -0.433, P < 0.0001), the percentage of free leptin (r = -0.732, P < 0.0001) and the absolute free leptin concentration (r = -0.506, P < 0.0001). The SLEPR level was positively correlated with the percentage of bound leptin (r = 0.730, P < 0.0001), whereas there was little variation in the absolute bound leptin concentration, regardless of the BMI or SLEPR concentration. During the IVF cycle, total and free leptin elevated during maximal ovarian stimulation, whereas there was no significant difference in the SLEPR concentration. CONCLUSIONS: The results demonstrate a skillful mechanism where a change in the serum SLEPR level regulates, in part, the biological activity of leptin in the circulation.     

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor γ chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor γ chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor γ chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

Immunohistochemical study of synphilin-1 in brains of patients with dementia with Lewy bodies - synphilin-1 is non-specifically implicated in the formation of different neuronal cytoskeletal inclusions

The caudal part of the fastigial nucleus, or the fastigial oculomotor region (FOR), plays an important role in executing accurate saccades. Inactivation of a monkey FOR leads to dysmetric saccades. Currently available data suggest that the dysmetria could be described as a parametric, uniform change in saccadic gain or, alternatively, as a constant error in the specification of the saccadic goal. To discriminate between these two possibilities, we examined the effect of FOR inactivation in the monkey. After a unilateral injection of muscimol into the FOR, ipsiversive saccades overshot a target. Gains were similar for movements of different sizes. The overshoot increased proportionately with the target distance and had a very small constant component. The present study indicates that the hypermetria of ipsiversive saccades after inactivation of the monkey FOR is primarily due to a uniform gain increase for all sizes of saccades.     

A comparison of three bioelectrical impedance analyses for predicting lean body mass in a population with a large difference in muscularity

This study tested the hypothesis that, as compared to whole-body bioelectrical impedance (BI) analysis, segmental BI analysis can estimate lean body mass (LBM) more accurately in a population with a large difference in muscularity. In addition to whole-body BI, which determines impedance (Z) between the wrist and ankle, two segmental BI analyses which determine the Z value of every body segment in each of (1) the arms, legs and trunk (distal BI) and (2) the upper arms, upper legs and trunk (proximal BI) were applied to a group of 125 male athletes and 75 non-athletes. The subjects were divided into validation and cross-validation groups. Simple and multiple regression analyses were applied to (length)²/Z (BI index) values for the whole-body and each body segment, to develop the prediction equations of LBM measured using air-displacement plethysmography. In the validation group, the SE of estimation was similar in the whole-body (3.4 kg, 5.4%), distal (3.4 kg, 5.5%) and proximal BI (3.3 kg, 5.2%) analyses. However, the whole-body and distal BI analyses produced systematical errors in the estimates of LBM. Moreover, the residuals in the two methods significantly (P<0.05) correlated with the ratios of BI indices of the upper arms and upper legs to those of the arms and legs, respectively, calculated as variables approximating the relative development of lean tissues at the proximal area of limbs. On the other hand, the proximal BI analysis was validated and cross-validated. Thus, the accuracy of estimating LBM was similar in the whole-body and the two segmental BI analyses. However, the prediction equations derived from the use of the whole-body BI index and a combination of the arms, legs and trunk BI indices produced a systematical error relating to the difference between the limb segments in lean tissue development.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.     

Dna ploidy of pheochromocytoma on cytology specimen by image analysis

Pheochromocytoma usually shows prominent nuclear atypia, but the presence of such atypical cells is known to be an unreliable predictor of malignancy. DNA ploidy of pheochromocytomas has been analyzed by flow cytometry or photospectrometry on paraffinem-bedded tissue, but the results were controversial. We performed DNA analysis on cytology specimens of 11 pheochromocytomas using an image analysis system. All tumors had a mixed pattern of a large population of diploid cells and a small population of polyploid cells. DNA content correlated with nuclear size, and larger cells had more DNA content. Such larger tumor cells had polyploid nuclei, such as 4 C, 8 C, 16 C, and 32 C, in both malignant and benign pheochromocytomas. The larger polyploid nuclei may result from difficulty of duplication at the mitotic phase of the cell cycle.