Monocyte chemotactic S19 ribosomal protein dimer in atherosclerotic vascular lesion.

To elucidate the molecular mechanism inducing monocyte/macrophage infiltration in the atherosclerotic lesion, we measured the monocyte chemotactic capacity in the extracts of aortic lesions. Five out of seven extracts exhibited significant chemotactic activities. Immunohistochemical examination with an anti-CD68 monoclonal antibody demonstrated that the five positive lesions possessed obvious monocyte/macrophage infiltrations at the intima, whereas the two negative lesions did so at significantly lower intensities. We subjected the chemotactic extracts to immunological analyses to identify the monocyte chemoattractant in them. The monocyte chemotactic capacities of all positive extracts were removed with anti-S19 ribosomal protein (RP S19) antibody beads and antimonocyte chemoattractant protein-1 (MCP-1) antibody beads. In three of the five extracts, the anti-RP S19 antibody beads were more effective than the anti-MCP-1 antibody beads for removal, while in the remaining two extracts, the opposite was observed. A combined immunoabsorption with these beads depleted the monocyte chemotactic capacity of a representative sample of each group. Consistently, the chemotactic capacity of an apparently RP S19 dimer-predominant extract was strongly inhibited by the presence of a C5a receptor antagonist. These results suggest that the RP S19 dimer and MCP-1 play a major role in the monocyte/macrophage infiltration of the atherosclerotic vascular lesion.

Vitellogenin detection and chick pathology are useful endpoints to evaluate endocrine-disrupting effects in avian one-generation reproduction study.

To investigate additional endpoints for screening of endocrine disruptors in birds, effects of 17beta-estradiol (E2) on one-generation reproduction in the Japanese quail (Coturnix japonica) were assessed. Pairs of the 10-week-old Japanese quail were fed a low-phytoestrogen diet containing E2 at 0 (control), 10, 100, and 1,000 ppm for six weeks. In the E2 100- and 1,000-ppm groups, the parental quail represented marked toxic changes including high mortality, decreased food consumption, decreased gonad weights, gross and histologic toxic changes in the reproductive and other organs, and inhibition of the reproduction. However, no adverse effects were observed in the parental quail from the E2 10-ppm group. In the parental males, serum vitellogenin (VTG) concentrations were increased significantly in the E2 10-ppm group, disclosing that serum VTG concentration is one of the highly sensitive endpoints for evaluating estrogenic endocrine activities. In the E2 10-ppm group, number of eggs laid, number of eggs with abnormalities, eggshell strength and thickness, fertility, early and late viabilities of embryos, normal hatchling rate, and clinical signs, mortality, viability, and body weight of chicks at 14 d of age were not affected. However, histopathology of the chicks in the E2 10-ppm group revealed meaningful morphological changes in the reproductive organs, such as cystic dilatation of seminiferous tubules, increased interstitial cells in the testis, and decreased theca cells in the ovary. The present study suggests that serum VTG concentration in the parental quail and histopathology of reproductive organs in the offspring are sensitive endpoints and are useful as additional endpoints in the avian one-generation reproduction test using the Japanese quail for evaluating estrogenic endocrine-disrupting effects.

Development of vitellogenin assay for endocrine disrupters using medaka (Oryzias latipes).

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (VTG) in the liver of medaka (Oryzias latipes) and was employed to establish an in vivo testing system for estrogen and estrogenic compounds using liver homogenates. Results of 3-month-old fish exposed to three reference chemicals (ethynylestradiol, methyltestosterone and flutamide) recommended by Organisation for Economic Co-operation and Development (OECD) for the validation showed the induction and decrease of vitellogenic responses, making the assay using the liver VTG of medaka a possible screening method for not only estrogens but also androgens. In addition, 21-day exposure of male fish to 4-tert-octylphenol and 4-nonylphenol produced concentration-dependent inductions of liver vitellogenin, with the lowest observed effect concentrations of 64.1 microg/L and 22.5 microg/L, respectively, while no significant VTG responses were observed in male and female fish exposed to tributyltin chloride and dibutyl phthalate. This study demonstrates that the VTG assay using liver homogenates from small fish such as medaka can be used as a screening method for environmental estrogens. This is because the sensitivity of the VTG response in medaka may be almost the same as that of other fish using blood samples.

[Establishment of an ELISA system of N1,N12-diacetylspermine in human urine].

OBJECTIVE: It is known that two kinds of Diacetylpolyamine, N1,N12-Diacetylspermine(DiAcSpm) and N1,N8-Diacetylspermidine(DiAcSpd), are excreted in urine. Although these two substances are 0.4% and 1.2% of the whole polyamine, respectively, these substances may be important for a sick diagnosis. The aim of this study was to develop a sensitive and specific method for detecting DiAcSpm. METHODS: We developed a new competitive ELISA system for the measurement of DiAcSpm in human urine, using polyclonal antibodies against DiAcSpm. RESULTS: The lower limit of detection of this ELISA was 4.53 nM/assay. The higher limit of detection was 145 nM/assay. Mean recovery was 101% (range, 96.3-108%). The coefficients of variation (CV) for within-run measurements by this method were 4.87% (mean = 95.5 nM) and 5.20% (mean = 32.4 nM), and the between-run CV were 7.53% (mean = 101 nM) and 9.46% (mean = 33.8 nM). CONCLUSIONS: We have established an ELISA system for the quantification of urinary DiAcSpm that uses novel polyclonal antibodies. Our ELISA system is simple and sensitive.

Generation of high-affinity antibody against T cell-dependent antigen in the Ganp gene-transgenic mouse.

Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (Ganp(Tg)). Ganp(Tg) displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (K(D) approximately 2.50 x 10(-7) M) of IgG1/lambda1 and contained the V(H)186.2 region with W33L mutation. Ganp(Tg) generated much higher affinity (K(D) > 1.57 x 10(-9) M) by usage of V(H)186.2 as well as noncanonical V(H)7183 regions. Ganp(Tg) also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (K(D) > 9.90 x 10(-11) M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.

S19 ribosomal protein dimer augments metal-induced apoptosis in a mouse fibroblastic cell line by ligation of the C5a receptor.

To analyze the role of S19 ribosomal protein (RP S19) in apoptosis, murine NIH3T3 were transfected with either hemagglutinin peptide-tagged (HA) wild-type human RP S19 or a mutant (Gln137Asn) that is resistant to transglutaminase-catalyzed cross-linked-dimerization. Transfection with the mutant HA-RP S19 inhibited manganese (II) (Mn II)-induced apoptosis whereas the wild-type HA-RP S19 augmented apoptosis and a mock transfection had no effect. Release of the wild-type HA-RP S19 dimer but not the mutant HA-RP S19 was observed during the apoptosis. The reduced rate of apoptosis of the cells transfected with the mutant HA-RP S19 was overcome by addition of extracellular wild-type RP S19 dimer. The apoptosis rates in cells transfected with either form of human HA-RP S19 and in mock transfectants were reduced to about 40% by the presence of anti-RP S19 antibody in the culture medium. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analysis showed that the cell surface expression of the receptor for cross-linked RP S19 dimer, C5a receptor, increased during apoptosis, concomitant with phosphatidylserine exposure. The expression of the C5a receptor gene also increased twofold. Apoptosis rates in the transfected and control cell lines were also reduced by the presence of an anti-mouse C5a receptor monoclonal antibody or of a peptide C5a receptor antagonist. These results indicated the presence of an RP S19 dimer- and C5a receptor-mediated autocrine-type augmentation mechanism during Mn II-induced apoptosis in the mouse fibroblastic cell line. In contrast to the RP S19 dimer, C5a actually inhibited apoptosis, suggesting that signaling through the C5a receptor varies depending on the ligand bound.

In situ evaluation of podocin in normal and glomerular diseases.

BACKGROUND: Mutations of the NPHS2 gene are responsible for autosomal-recessive steroid-resistant nephrotic syndrome. Its product, podocin, faces the slit diaphragm area with its two ends in the cytoplasm of foot processes. METHODS: We generated rabbit polyclonal antibodies against conjugated peptides from human podocin N- and C-termini, and studied podocin and synaptopodin using kidney tissues of normal humans and those with glomerular diseases. RESULTS: Antipodocin antibodies detected the original 42 kD fragment and an extra smaller fragment by Western blot analysis using human isolated mature glomeruli. RNA analysis showed two bands, the original and the other of a decreased length. Immunohistochemically, podocin was detected in a linear pattern along the glomerular capillary loop. Antipodocin antibody (C-terminal) stained the smooth muscles of renal arterioles and aorta. Among 42 patients, podocin was normally expressed in glomeruli in purpura nephritis, IgA nephropathy (IgAN), and minimal-change disease (MCD), while it was either decreased or absent in most subjects with focal segmental glomerulosclerosis (FSGS). The expression of synaptopodin was similar to that of podocin, although some discrepancy existed. CONCLUSION: Although indirect, our data suggest the existence of a vascular isoform of podocin with a different molecular mass. We propose that examination of podocin expression may help differentiate MCD from FSGS.