Screening for lectin-like protein-producing microorganisms based on cell surface proteins

A method for screening lectin-producing microorganisms was developed. The presence of lectin on microbial cell surfaces was used as an index for their selective isolation. The lectin-producing microorganisms adhered to sugar-modified agarose beads and were selectively eluted with specific saccharide solutions. Spin columns were an effective tool for excluding non-lectin producers. Eighty-seven percent of the microorganisms that were eluted from the beads showed hemagglutination. The results of sequence analysis indicated that some of the eluted microorganisms have not been previously identified as lectin-producing microorganisms.     

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S₁ layer at the early S₁ formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S₁ layer, but showed uneven distribution in the S₂ layer. AGX labeling was mainly detected in the inner S₂ layer after the beginning of the helical cavity formation. The outer S₂ layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S₂ layer. These patterns of AGX labeling in the S₂ layer opposed the lignin and β-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S₂ layer from the early S₂ formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.     

Genetic characteristics and antimicrobial resistance of Escherichia coli from Japanese macaques (Macaca fuscata) in rural Japan

Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to investigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the wild macaque population in Shimokita Peninsula, a rural area of Japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 for E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and microbial sensitivity tests. In total, 248 E. coli strains were isolated from 159 fecal samples from wild macaques, and 42 E. coli were isolated from 17 samples from captive macaques. None of the virulence genes eae, stx, elt, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling. Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates had no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was <70%. No plasmid profile was shared between wild-derived and captive-derived isolates. The prevalence of antimicrobial-resistant E. coli was 6.5% (n=62) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.     

Ca influx and ATP release mediated by mechanical stretch in human lung fibroblasts

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca²⁺]_i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca²⁺]_i. The stretch-induced [Ca²⁺]_i elevation was attenuated in Ca²⁺-free solution. In contrast, the increase of [Ca²⁺]_i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd³⁺, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca²⁺]_i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca²⁺ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.     

Dissection of the role of p62/Sqstm1 in activation of Nrf2 during xenophagy

Upon infection of a cell by Salmonella, p62/Sqstm1 assembles on the microbes; simultaneously, p62/Sqstm1 is phosphorylated at Ser351, leading to inactivation of Keap1, which is responsible for degrading Nrf2. Thus, cytoprotective Nrf2 targets are induced at the same time that autophagosomes entrap the microbes (xenophagy). However, the detailed role of p62/Sqstm1 during xenophagy has remained unclear. Here we show that translocation of p62/Sqstm1 to invasive Salmonella precedes Ser351 phosphorylation. Furthermore, in addition to Ser351 phosphorylation, oligomerization of p62/Sqstm1 is also required for localization of Keap1 onto microbes, which is followed by Nrf2 activation. Our data reveal the sequential dynamics of p62/Sqstm1 in response to bacterial infection.     

Inhibition of transforming growth factor-β signaling potentiates tumor cell invasion into collagen matrix induced by fibroblast-derived hepatocyte growth factor

Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial‐mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor–stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.     

Specific Targeting of a Naturally Presented Osteosarcoma Antigen,Papillomavirus Binding Factor Peptide,Using an Artificial Monoclonal Antibody

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (K_D = 1.53 × 10⁻⁹ m) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.     

ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that ΔNp63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and in vitro studies were also performed to examine the functional relevance of ΔNp63 in the expression of TSLP receptors and the constituting autocrine and/or paracrine pathway of TSLP under the condition of stimuli to innate receptors sensing cell damage. The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked ΔNp63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of ΔNp63 (ΔNp63^lo/-). Moreover, in the absence of ΔNp63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without ΔNp63, the results indicate that ΔNp63^lo/- keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.