cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.     

Schizophrenia, eye movements, and biocultural heterogeneity

Smooth-pursuit eye-tracking dysfunction is a putative genetic trait marker for schizophrenia. In this study 88 Japanese schizophrenics from Kyushu and Okinawa were examined for the marker using precise high-resolution instrumentation: 76% of the schizophrenics from Kyushu and 89% of those from Okinawa had pursuit dysfunction. The presence of the culture-neutral smooth-pursuit marker for schizophrenia in Japan demonstrates that the etic concept "schizophrenia" is cross-culturally valid. Furthermore, the ubiquity of the marker in biologically and culturally diverse populations may indicate a limit on the extent of meaningful heterogeneity likely to be discovered within the condition.     

Molecular cloning and characterization of mouse Tspan-3, a novel member of the tetraspanin superfamily, expressed on resting dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells and play an essential role for triggering T-cell-mediated immune responses. In search for novel cell surface molecules expressed on DCs involved in T cell priming by representational differential analysis, we identified a mouse homologue of Tspan-3 (mTspan-3), a novel member of the tetraspanin superfamily. The mTspan-3 consists of four hydrophobic, putative transmembrane regions, forming a small and a large extracellular loop, with short intracellular amino and carboxil tails. Although the mTspan-3 is expressed on a variety of immune cell types including resting DCs, its expression on DCs is downregulated during activation induced by cross-linking CD40 with anti-CD40 monoclonal antibody. These results suggest that mTspan-3 may be involved in the function of DCs in association with T cell stimulation.     

Interactions among yeast protein-disulfide isomerase proteins and endoplasmic reticulum chaperone proteins influence their activities

We previously reported that the reductive activities of yeast protein-disulfide isomerase (PDI) family proteins did not completely explain their contribution to the viability of Saccharomyces cerevisiae (Kimura, T., Hosoda, Y., Kitamura, Y., Nakamura, H., Horibe, T., and Kikuchi, M. (2004) Biochem. Biophys. Res. Commun. 320, 359-365). In this study, we examined oxidative refolding activities and found that Mpd1p, Mpd2, and Eug1p exhibit activities of 13.8, 16.0, and 2.16%, respectively, compared with Pdi1p and that activity for Eps1p is undetectable. In analyses of interactions between yeast PDI proteins and endoplasmic reticulum molecular chaperones, we found that Mpd1p alone does not have chaperone activity but that it interacts with and inhibits the chaperone activity of Cne1p, a homologue of mammalian calnexin, and that Cne1p increases the reductive activity of Mpd1p. These results suggest that the interface between Mpd1p and Cne1p is near the peptide-binding site of Cne1p. In addition, Eps1p interacts with Pdi1p, Eug1p, Mpd1p, and Kar2p with dissociation constants (KD) in the range of 10(-7) to 10(-6). Interestingly, co-chaperone activities were completely suppressed in Eps1p-Pdi1p and Eps1p-Mpd1p complexes, although only Eps1p and Pdi1p have chaperone activity. The in vivo consequences of these results are discussed.     

Stopped-flow investigation of the reaction between vitamin E radical and vitamin C in solution

Kinetic study of the reaction between vitamin E radical and vitamin C has been performed. The rates of reaction of vitamin C (ascorbic acid 1, 6-0-stearyl ascorbic acid 2, and 2,6-O-dipalmitoyl ascorbic acid 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl) in benzene-ethanol (2:1, v/v) solution have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants obtained are 549 +/- 30 M-1s-1 for 1, 626 +/- 53 M-1s-1 for 2, and 4.84 +/- 1.41 M-1s-1 for 3 at 25.0 degrees C. The result shows that the ascorbic acid ester 2 having a long-alkyl-chain at 6-position is 1.14 times as reactive as the ascorbic acid 1, whereas the ascorbic acid ester 3 substituted at 2-position is only 0.01 times as reactive as the ascorbic acid 1.     

Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.     

Sequence rearrangement in JC virus DNAs molecularly cloned from immunosuppressed renal transplant patients.

From nonimmunocompromised individuals, we have recently identified a possible archetypal JC virus DNA sequence from which various regulatory sequences of JC virus isolates derived from patients with progressive multifocal leukoencephalopathy (PML) could have evolved. In this study, we analyzed the regulatory sequences of JCV DNAs cloned from urine samples of a PML risk group (renal transplant patients on immunosuppressive therapy). A number of JC virus DNAs were molecularly cloned from virions excreted in the urine of eight patients. Furthermore, fragments containing the regulatory region were amplified by the polymerase chain reaction and subsequently molecularly cloned from cell-associated JC virus excreted in the urine of two patients. The regulatory regions in all clones were analyzed with restriction enzymes, and those in representative clones were sequenced. We found that clones with the archetypal regulatory sequence were predominant in all urine samples, but a few clones carried regulatory sequences that diverged from the archetypal sequence by deletion or duplication. The finding that sequence rearrangement in the archetypal regulatory region occurs in the course of infection in immunosuppressed hosts is consistent with the adaptation hypothesis which has been put forward to explain the divergence of the regulatory regions in PML-derived JC virus isolates.