Comparison of enzymatic properties between hPADI2 and hPADI4

In the sera of rheumatoid arthritis (RA) patients, autoantibodies directed to citrullinated proteins are found with high specificity for RA. Peptidylarginine deiminases (PADIs) are enzymes responsible for protein citrullination. Among many isoforms of PADIs, only PADI4 has been identified as an RA-susceptibility gene. To understand the mechanisms of the initiation and progression of RA, we compared the properties of two PADIs, human PADI2 and human PADI4, which are present in the synovial tissues of RA patients. We confirmed their precise distribution in the RA synovium and compared the stability, Ca2+ dependency, optimal pH range, and substrate specificity. Small but significant differences were found in the above-mentioned properties between hPADI2 and hPADI4. Using LC/MS/MS analysis, we identified the sequences in human fibrinogen indicating that hPADI2 and hPADI4 citrullinate in different manners. Our results indicate that hPADI2 and hPADI4 have different roles under physiological and pathological conditions. Further studies are needed for the better understanding of the role of hPADIs in the initiation and progression of RA.     

Inhibition of antithrombin by hyaluronic acid may be involved in the pathogenesis of rheumatoid arthritis

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, proinflammatory processes, and proliferation of fibroblast-like cells. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. Tissue destruction in joints causes the accumulation of large quantities of free hyaluronic acid (HA) in RA synovial fluid. The present study was conducted to investigate the effects of HA and several other glycosaminoglycans on antithrombin, a plasma inhibitor of thrombin. Various glycosaminoglycans, including HA, chondroitin sulfate, keratan sulfate, heparin, and heparan, were incubated with human antithrombin III in vitro. The residual activity of antithrombin was determined using a thrombin-specific chromogenic assay. HA concentrations ranging from 250 to 1000 μg/ml significantly blocked the ability of antithrombin to inhibit thrombin in the presence of Ca²⁺ or Fe³⁺, and chondroitin A, B and C also reduced this ability under the same conditions but to a lesser extent. Our study suggests that the high concentration of free HA in RA synovium may block antithrombin locally, thereby deregulating thrombin activity to drive the pathogenic process of RA under physiological conditions. The study also helps to explain why RA occurs and develops in joint tissue, because the inflamed RA synovium is uniquely rich in free HA along with extracellular matrix degeneration. Our findings are consistent with those of others regarding increased coagulation activity in RA synovium.     

Characterization of histopathology and gene-expression profiles of synovitis in early rheumatoid arthritis using targeted biopsy specimens

The disease category of early rheumatoid arthritis (RA) has been limited with respect to clinical criteria. Pathological manifestations of synovitis in patients whose disease is clinically classified as early RA seem to be heterogeneous, with regular variations. To clarify the relation between the molecular and histopathological features of the synovitis, we analyzed gene-expression profiles in the synovial lining tissues to correlate them with histopathological features. Synovial tissues were obtained from knee joints of 12 patients with early RA by targeted biopsy under arthroscopy. Surgical specimens of long-standing RA (from four patients) were examined as positive controls. Each histopathological parameter characteristic of rheumatoid synovitis in synovial tissues was scored under light microscopy. Total RNAs from synovial lining tissues were obtained from the specimens selected by laser capture microdissection and the mRNAs were amplified by bacteriophage T7 RNA polymerase. Their cDNAs were analyzed in a cDNA microarray with 23,040 cDNAs, and the levels of gene expression in multilayered lining tissues, compared with those of normal-like lining tissues in specimens from the same person, were determined to estimate gene-expression profiles characteristic of the synovial proliferative lesions in each case. Based on cluster analysis of all cases, gene-expression profiles in the lesions in early RA fell into two groups. The groups had different expression levels of genes critical for proliferative inflammation, including those encoding cytokines, adhesion molecules, and extracellular matrices. One group resembled synovitis in long-standing RA and had high scores for some histopathological features – involving accumulations of lymphocytes and plasma cells – but not for other features. Possible differences in the histopathogenesis and prognosis of synovitis between the two groups are discussed in relation to the candidate genes and histopathology.     

Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.     

Physical and functional interactions of the lysophosphatidic acid receptors with PDZ domain-containing Rho guanine nucleotide exchange factors (RhoGEFs)

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRs) including LPA1, LPA2, and LPA3. LPA-induced cytoskeletal changes are mediated by Rho family small GTPases, such as RhoA, Rac1, and Cdc42. One of these small GTPases, RhoA, may be activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors (RhoGEFs) under LPA stimulation although the detailed mechanisms are poorly understood. Here, we show that the C terminus of LPA1 and LPA2 but not LPA3 interact with the PDZ domains of PDZ domain-containing RhoGEFs, PDZ-RhoGEF, and LARG, which are comprised of PDZ, RGS, Dbl homology (DH), and pleckstrin homology (PH) domains. In LPA1- and LPA2-transfected HEK293 cells, LPA-induced RhoA activation was observed although the C terminus of LPA1 and LPA2 mutants, which failed to interact with the PDZ domains, did not cause LPA-induced RhoA activation. Furthermore, overexpression of the PDZ domains of PDZ domain-containing RhoGEFs served as dominant negative mutants for LPA-induced RhoA activation. Taken together, these results indicate that formation of the LPA receptor/PDZ domain-containing RhoGEF complex plays a pivotal role in LPA-induced RhoA activation.     

Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.     

RA-RhoGAP, Rap-activated Rho GTPase-activating protein implicated in neurite outgrowth through Rho

Rap1 and Rho small G proteins have been implicated in the neurite outgrowth, but the functional relationship between Rap1 and Rho in the neurite outgrowth remains to be established. Here we identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 in the neurite outgrowth. RA-RhoGAP has the RA and GAP domains and showed GAP activity specific for Rho, which was enhanced by the binding of the GTP-bound active form of Rap1 to the RA domain. Overexpression of RA-RhoGAP induced inactivation of Rho for promoting the neurite outgrowth in a Rap1-dependent manner. Knockdown of RA-RhoGAP reduced the Rap1-induced neurite outgrowth. These results indicate that RA-RhoGAP transduces a signal from Rap1 to Rho and regulates the neurite outgrowth.     

Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae)

Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean Mw 2 x 10(4) and 1.15 x 10(6)Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.     

Effects of free proline accumulation in petunias under drought stress

Petunias (Petunia hybrida cv. 'Mitchell') accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by Delta(1)-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57-1.01% of the total amino acids in the transgenic plants, or 1.5-2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.     

Air-liquid interface promotes invasive growth of laryngeal squamous cell carcinoma with or without hypoxia

Air-liquid interface (ALI) is a microenvironment of aerodigestive tract. In our previous study, ALI promoted invasive growth of laryngeal squamous cell carcinoma (SCC); but its mechanism was unclear. Hypoxia is also related to cancer spread. Here we show that ALI with or without hypoxia accelerated invasive growth of laryngeal SCC cells, using collagen gel invasion assay. Submerged condition (SMC) without ALI did not induce the invasion with or without hypoxia. ALI enhanced expression of the following growth-, invasion-, and motility-related molecules in the cells with or without hypoxia more greatly than SMC: c-Met, Ras, mitogen-activated protein kinase cascade proteins (Raf-1, MEK-1, and ERK-1/2), matrix metalloproteinase-1, and filamin A. The data indicate that ALI promotes invasive growth of SCC cells by enhancement of the invasive growth-related molecules above, through mechanisms that differ from hypoxia, suggesting that ALI microenvironment should be taken into account for the study of cancer biology.