Characterization of catalytic α-1,3-glucanase isozymes from Paenibacillus glycanilyticus FH11 by using Brevibacillus system; Essential for suppression of Streptococcus mutans biofilms

S. mutans has been implicated in the etiology of dental caries by facilizing the colonization of tooth surfaces and playing a key role in the development of the virulent dental plaque. α-1,3-Glucan, which is a key structural constituent of the biofilm matrix (dental plaque), synthesized by glucosyltransferase type B (gtfB) in the presence of ingested sucrose. α-1,3-Glucanases also called mutanases, which hydrolyze α-1,3-glucan, are classified into two families of glycoside hydrolases, fungal (type 71) and bacterial (type 87). Because of being considered to degrade α-1,3-glucan, α-1,3-glucanases have been purified and characterized from various microbial sources. However, there are few reports on S. mutans biofilm study. For the host cell expression, Brevibacillus system is an effective bacterial expression system for secretory proteins. B. choshinensis is a gram-positive bacterium and easy to handle non-sporulating bacterium, lacking extracellular protease, that has been already shown to provide a high level of recombinant protein expression. Recently, many proteins are produced from this expression system and use for medical treatment, research study (1). Therefore, in this study we attempted to use Brevibacillus expression system to express, purify, and characterize of α-1,3- glucanase. In addition, we aimed to investigate the effect of recombinant enzyme on α-1,3-glucan biofilm produced by S. mutans from the viewpoints of formation and the effect of toothpaste agent on enzyme activity. Two novel catalytic domains of α-1,3-glucanase isozyme genes were cloned from P. glycanilyticus strain FH11 and heterologously expressed in Brevibacillus system. The recombinant isozymes, in termed CatAgl-FH1 and CatAgl-FH2, were purified to homogeneity with specific activity 0.70 U/mg and 0.77 U/mg respectively. The molecular mass of catalytic domain was estimated 62 kDa by SDS-PAGE. Both recombinant enzymes exhibited the different properties. The optimal pH of CatAgl-FH1 and CatAgl-FH2 were 5.5 and 6.0, respectively. The pH stability of CatAgl-FH1 and CatAgl-FH2 were in a range of pH 4.0-11.0 and 4.5-9.0, respectively. The optimal temperature of CatAgl-FH1 and CatAgl-FH2 were 60°C and 55°C, respectively and they were stable until 60°C. Thin Layer chromatography revealed their mode of hydrolysis towards α-1,3-glucan was endo-cleavage pattern. The major products of CatAgl-FH1 were di- and trisaccharide but mainly trisaccharide was for CatAgl-FH2. Both enzymes showed high tolerance against high concentration of sodium fluoride. However, each enzyme activity on surfactants were stepped down when sodium dodecyl sulfate and benzethonium concentration were increased. Please click Additional Files below to see the full abstract

Expressions of Vascular Endothelial Growth Factor (VEGF)-D and VEGF Receptor-3 in Colorectal Cancer: Relationship to Lymph Node Metastasis

Angiogenic factors play a major role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF)- D is a ligand for VEGF receptor-3 (VEGFR-3/Flt-4), which mainly expressed on the lymphatic endothelium. Recent experimental studies have shown that VEGF-D induces tumor lymphangiogenesis and promote metastatic spread of tumor cells via lymphatic vessels. However, the contribution of VEGFD to lymph node metastasis in human colorectal cancer is less understood. We therefore examined VEGF-D and VEGFR-3 expression in patients with colorectal cancer. Sections of formalin-fixed and paraffin-embedded specimens from 76 colorectal cancers were immunohistochemically stained for VEGF-D and VEGFR-3. Staining for VEGF-D was positive in the cytoplasm of tumor cells in 43 of 76 examined tumors (56.6%). Staining for VEGFR-3 was positive in endothelial cells in 38 (50.0%) tumors. Univariate analysis showed that both VEGF-D and VEGFR-3 expressions correlated significantly with lymph node metastasis, histological type and depth of tumor invasion. However, logistic regression analysis indicated that VEGF-D expression, but not that of VEGFR-3, was an independent predictor for lymph node metastasis. Our data suggest that VEGF-D plays an important role in lymph node metastasis in colorectal cancer

Elevated Expression of Poly(ADP-Ribose) Polymerase-1 is Associated with Liver Metastasis in Colorectal Cancer.

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) and its subsequent cleavage are early markers of apoptosis. PARP-1 is associated with DNA repair and so chromosome stability, cell cycle regulation, as well as tumorigenesis. To investigate the role of PARP-1 expression in colorectal carcinoma and its metastasis of liver, we compared the expression of PARP-1 in primary colorectal cancers with (n=15) and without liver metastasis (n=17) using a semi-quantitative reverse transcription - polymerase chain reaction. We also examined the expressions of poly(ADP-ribose) (PAR) and p53 in these tumors by immunohistochemistry. A significantly higher PARP-1 mRNA expression was noted in tumors with liver metastasis than those without liver metastasis (p<0.01). Colorectal cancers positively stained for p53 exhibited significantly higher PARP-1 mRNA expression than p53-negative tumors (p<0.01). The PAR labeling index (LI) of tumors with metastasis (0.33 ﾂｱ 0.33) was not significantly different (p=0.35) from that of tumors without liver metastasis (0.38 ﾂｱ 0.19). p53-positive tumors tended to have higher PAR LI levels than p53-negative tumors (p=0.08). Our findings suggest that PARP-1 may contribute to liver metastasis due to its DNA repair activity, resulting in survival of the tumor cells with accumulation of metastaticrelated gene\u27s damages. Detailed analysis of PARP-1 may be useful in cancer research and/or cancer therapy

Liquid-crystalline phase transition in organophosphazenes

Organophosphazenes with a similar mesogenic moiety were prepared and their mesogenicity was studied by differential scanning calorinetry (DSC) measurements and polarizing microsope observations. In cyclotriphosphazenes with alk-­oxybiphenyl and Schiff base moieties, mesomorphic phase transitions were observed, but no mesomorphic phase was observed for the corresponding cyclotetraphosphazenes. In polyphos-­phazenes with an alkoxybiphenyl moiety, no mesomorphic phase was observed. The molecular structure of cyclotriphosphazenes facilitated the formation of a mesomorphic layer structure; in contrast, the formation of a mesomorphic layer structure did not occur in cyclotetraphosphazenes and polyphosphazenes, even though they bore a similar mesogenic moiety. Moreover, in cyclotriphosphazenes with an optically active alkoxybiphenyl group, a smectic C* phase was observed. The spontaneous polarization of the compound was −190 μ C m −2 at 436 K in 25 μ in cell using the triangular-wave method. © 1998 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38314/1/785_ftp.pd

Clinicopathology and prognosis of mucinous gastric carcinoma

Background/Aims: Mucinous gastric carcinoma (MGC) is a rare histopathological type of gastric carcinoma, for which the clinicopathological features and prognosis remain controversial. To clarify the clinical significance of mucinous histological type in gastric cancer, we studied clinicopathological characteristics of MGC tumors and prognosis of patients. Methodology: Forty-one patients with MGC and 1,407 patients with non-mucinous gastric carcinoma (NGC) were included in the study. Tumors were evaluated against patient gender and age, tumor location, size, and macroscopic type, depth of gastric wall invasion, lymph node metastasis, liver metastasis, peritoneal dissemination, distant metastasis, stage, and operative curability. Results: Compared with NGC tumors, MGC tumors were larger, showed more serosal invasion, were associated with a higher incidence of lymph node metastasis, and peritoneal dissemination, and tended to be at a more advanced stage. However, multivariate analysis demonstrated that the mucinous histological type was neither an independent prognostic factor nor an independent risk factor for lymph node metastasis in patients with gastric cancer. Conclusions: The mucinous histological type had no influence on patient outcome or the frequency of lymph node metastasis. MGC tumors are therefore biologically similar to those in NGC

Molecular Mass and Localization of α-1,3-Glucan in Cell Wall Control the Degree of Hyphal Aggregation in Liquid Culture of Aspergillus nidulans

α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE) or agsB (agsBOE), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE, 41.6 ± 5.8 kDa; agsBOE, 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain–fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans

High Level Expression of Platelet-derived Endothelial Cell Growth Factor predicts Good Prognosis in Colorectal Cancer Patients

Platelet-derived endothelial cell growth factor (PD-ECGF) is one of the angiogenic factors. PD-ECGF expression is elevated in colorectal carcinoma, but its prognostic value does not reach a consensus. The aim of this study is to clarify the prognostic value of the PD-ECGF expression in colorectal carcinomas. PD-ECGF expression was measured by enzyme- linked immunosorbent assay in frozen materials from 71 colorectal cancer patients who had recived curative resection. Patients were divided into high expression and low expression groups based on cut-off value. Correlations among PD-ECGF expression, clinicopathologic features, and disease-free interval were studied by univariate and multivariate analysis. To evaluate the origin of PD-ECGF, serial sections of 71 tumors were stained for PD-ECGF and CD68. PD-ECGF expression in normal mucosa was 34.4 ﾂｱ 15.5 (Units/mg protein) and the cut-off value was 65.4 (mean+2SD). There were no significant correlations between clinicopathological features and PD-ECGF expression. The disease-free interval for high PD-ECGF expression group was significantly longer than that of low expression group (P=0.006). A multivariate Cox\u27s regression analysis revealed a high PD-ECGF expression as an independent factor for better outcome. In immunohistochemical study, almost all tumor cells were negative for PD-ECGF, but stromal macrophages were predominantly positive for PD-ECGF. In conclusion, the PD-ECGF expression measured in this study was derived from stromal macrophages. High PD-ECGF expression was a predictor for favorable outcome after curative resection for colorectal cancer

Understanding the Role of Temperature and Drain Current Stress in InSnZnO TFTs with Various Active Layer Thicknesses

Thin-film transistor (TFT) devices composed of metal oxide semiconductors have attracted tremendous research attention globally in recent years. Owing to their ability to offer mobility, metal oxide semiconductor materials can enable high-performance TFTs for next-generation integrated display devices. Nevertheless, further breakthroughs of metal oxide TFTs are mainly obstructed by their long-term variability, the reason for which is not yet fully understood. Herein, TFTs based on InSnZnO (ITZO) with various thicknesses (TITZO) were prepared and their long-term stabilities under test temperatures and drain current stress were investigated. The results indicate that ITZO TFTs exhibit outstanding electrical properties regardless of the TITZO, including a high saturated mobility of over 35 cm2V&minus;1s&minus;1 and sharp subthreshold swing. Note that the transfer and output characteristic curves of the device with a thick TITZO of 100 nm express an abnormal current surge when high gate and drain voltages are exerted, which is attributed to the floating body effect, caused when the imposed electric field induces impact ionization near the drain side. More interestingly, these drain current stress results further suggest that the abnormal shift behavior of the electrical properties of the ITZO TFTs with a TITZO of greater than 75 nm is observed to deteriorate gradually with increasing temperature and drain current bias. This study addresses that such a degradation effect should be restrained for the operation of high-mobility devices