Activation of a signaling cascade by cytoskeleton stretch

Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.     

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.0 ng/ml HGF neither formed a continuous basement membrane on collagen fibrils nor maintained a continuous basement membrane architecture on a basement membrane substratum. The cells showed increased secretion of matrix metalloproteinase-9 and urokinase-type plasminogen activator, and the HGF-induced inhibition of basement membrane formation was attenuated by addition of 200 ng/ml tissue inhibitor of matrix metalloproteinase-1. Cells sequentially exposed to HGF and 1.0 ng/ml transforming growth factor-beta1 had enhanced basement membrane formation compared with those receiving these reagents in the reverse order or concurrently. HGF simultaneously stimulated proliferation and migration of the cells so that it advanced wound closure on the basement membrane substratum. The present results indicate that the role of HGF in wound healing is the stimulation of reepithelization, but this factor may also contribute to the degradation of the basement membrane.     

Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.     

Horizontal Transmission of Bursaphelenchus xylophilus between Sexes of Monochamus alternatus

Four experiments were conducted using nematode-infested and nematode-free adults of the cerambycid beetle, Monochamus alternatus, to determine horizontal transmission pathways of Bursaphelenchus xylophilus. When nematode-infested beetles of one sex and nematode-free beetles of the opposite sex were paired in containers for 48 or 72 hours, the number of nematodes carried by nematode-free beetles tended to increase with increased number of nematodes carried by nematode-infested beetles. The nematodes acquired by "nematode-free" beetles could be transmitted to pine. A female beetle that received 13 nematodes from a male transmitted one nematode to a Pinus densiflora bolt via an oviposition wound. When the nematode-infested and nematode-free beetles were observed continuously, it was observed that the number of nematodes carried by nematode-free beetles at the end of the first sexual mounting increased as the number of nematodes carried by nematode-infested beetles just before mounting increased. The number of nematodes transferred to nematode-free beetles was positively related to duration time of mounting. There was no difference in transmission efficacy between male-to-female transmission and female-to-male transmission. The horizontal transmission pathways are discussed relative to the persistence of B. xylophilus in resistant pine forests and the control of pine wilt disease.     

Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Exocytotic "constipation" is a mechanism of tubulin/lysosomal interaction in colchicine myopathy

Colchicine, a known microtubule disrupting agent, produces a human myopathy, characterized by accumulation of lysosomes. We have created a reliable animal model of colchicine myopathy that replicates the subacute myopathy seen in humans, reproducing the chronic proximal weakness and vacuolar changes in nonnecrotic myofibers. If a microtubule network plays a role in lysosomal function in muscle, disturbance of it could alter degradation of intrinsic membrane receptors, presumably at some intracellular processing site or at exocytosis. Thus, we examined, as a possible cellular pathogenesis of colchicine myopathy, how the muscle cytoskeleton affects the degradation of membrane proteins, which are processed through the endosomal/lysosomal pathway. We used the acetylcholine receptor as a model membrane component in cultured myotubes allowed to preincubate with colchicine. We tested at which step colchicine interferes with receptor trafficking by accounting for internalization, delivery to lysosomes, hydrolysis, or exocytotic release of debris. We report that colchicine significantly decreases the exocytosis of AChRs but does not affect receptor internalization, lysosomal hydrolysis, or the number of surface membrane receptors. Further, our immunofluorescence observations revealed a morphologic tubulin network in rat skeletal muscle that is more densely distributed in white (mitochondria-poor) muscle fibers than in red (mitochondria-rich) fibers but is present in both. Ultrastructurally, immunogold labeling localized tubulin in the intermyofibrillar region in a long and linear fashion, unassociated with myofibers or mitochondria. Taken together, our findings suggest the following: (1) Microtubules likely play a functional role in the pathway of lysosomal degradation in normal adult skeletal muscle; (2) The observed decrease in overall apparent degradation of membrane receptors by colchicine must be due primarily to inhibition of exocytosis. These data indicate that lysosomal "constipation" underlies colchicine myopathy. (3) An animal model faithful to the human disorder will allow further pathogenetic studies.     

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol-chloroform-petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4-position of the non-reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF-kappaB activation in murine cells via Toll-like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4-dependent pathway similar to E. coli-type lipid A, even though these have structural differences.