Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

The increased invasiveness of gastric adenocarcinoma is important for progression and metastasis. In recent molecular biological studies, ribophorine II (RPN2) induced epithelial-mesenchymal transition and metastatic activity. However, no studies have evaluated the relationship between RPN2 expression, ability of cancer to invade/metastasis, and patient prognosis in gastric adenocarcinoma. Therefore, we have examined these factors. Immunohistochemical staining was performed to detect RPN2 and p53 in the primary lesion and adjacent normal gastric mucosa of 242 gastric adenocarcinoma patients who underwent resection surgery. We conducted clinicopathologic examinations and analyzed patient prognoses with the Kaplan-Meier method. Further, multivariate analysis was conducted using a Cox hazard model. Also, we analyzed the ability of invasion under inhibited RPN2 expression in vitro. RPN2 expression was observed in 119 of 242 cases of gastric adenocarcinoma patients. RPN2 expression was associated with a higher incidence of depth of wall invasion, lymph node metastasis, lymphatic invasion, venous invasion, peritoneal dissemination, histopathological stage, and p53 expression. In stage II and III curative resection cases, where recurrence is the most serious problem, cases that expressed RPN2 had a significantly lower 5-year survival rate and higher recurrence rate compared to the cases with no RPN2 expression. In the multivariate analysis for prognosis, RPN2 expression was found to be an independent factor. Also, gastric adenocarcinoma cell, had mutant-type p53, reduced the ability of invasion by knockout of RPN2 expression in vitro. RPN2 expression correlates with gastric adenocarcinoma cell invasion and shows promise as a new prognostic factor in human gastric adenocarcinoma.

Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight.

Levan is a fructan polymer that offers a variety of applications in the chemical, health, cosmetic and food industries. Most of the levan applications depend on levan molecular weight, which in turn depends on the source of the synthesizing enzyme and/or on reaction conditions. Here we demonstrate that in the particular case of levansucrase from Bacillus subtilis 168, enzyme concentration is also a factor defining the molecular weight levan distribution. While a bimodal distribution has been reported at the usual enzyme concentrations (1 U/ml equivalent to 0.1 µM levansucrase) we found that a low molecular weight normal distribution is solely obtained al high enzyme concentrations (>5 U/ml equivalent to 0.5 µM levansucrase) while a high normal molecular weight distribution is synthesized at low enzyme doses (0.1 U/ml equivalent to 0.01 µM of levansucrase).

Sucrose substitutes affect the cariogenic potential of Streptococcus mutans biofilms.

Streptococcus mutans is considered the primary etiologic agent of dental caries and contributes significantly to the virulence of dental plaque, especially in the presence of sucrose. To avoid the role of sucrose on the virulence factors of S. mutans, sugar substitutes are commonly consumed because they lead to lower or no production of acids and interfere with biofilm formation. This study aimed to investigate the contribution of sugar substitutes in the cariogenic potential of S. mutans biofilms. Thus, in the presence of sucrose, glucose, sucralose and sorbitol, the biofilm mass was quantified up to 96 h, the pH of the spent culture media was measured, the expression of biofilm-related genes was determined, and demineralization challenge experiments were conduct in enamel fragments. The presence of sugars or sugar substitutes profoundly affected the expression of spaP, gtfB, gtfC, gbpB, ftf, vicR and vicX in either biofilm or planktonic cells. The substitution of sucrose induced a down-regulation of most genes involved in sucrose-dependent colonization in biofilm cells. When the ratio between the expression of biofilm and planktonic cells was considered, most of those genes were down-regulated in biofilm cells in the presence of sugars and up-regulated in the presence of sugar substitutes. However, sucralose but not sorbitol fulfilled the purpose of reducing the cariogenic potential of the diet since it induced the biofilm formation with the lowest biomass, did not change the pH of the medium and led to the lowest lesion depth in the cariogenic challenge.

Prevalence of salivary Streptococcus mutans serotype k in children undergoing congenital heart surgery.

OBJECTIVE: The prevalence of Streptococcus mutans serotype k, which was speculated that might be associated with the development of cardiovascular diseases, has been reported in adult cardiovascular surgery patients. There is no information about presence of serotype k in children with cardiac disease. The aim of this study was to determine the salivary prevalence of S. mutans serotype k in children with congenital heart disease. STUDY DESIGN: Salivary samples of 25 patients undergoing elective surgery for congenital heart defects with cardiopulmonary bypass and an age and gender matched control group of 25 healthy children were enrolled in the study. Species-specific 16SrRNA gene sequences were used for S. mutans and serotype-specific rgpF gene sequences were used for S. mutans serotype k determination in stimulated saliva samples. RESULTS: S. mutans was detected in 19 (76%) of the study and 15 (60%) of the control children. The difference was not shown to be statistically significant. Serotype k was determined from 3 (12%) of the study group, while it was not determined from the samples of the control group. CONCLUSIONS: Our results indicate that those children with congenital heart disease may possess S. mutans serotype k in oral cavity at a higher frequency as similar with the adult cardiac surgery patients.

The new molecular markers DDIT3, STT3A, ARG2 and FAM129A are not useful in diagnosing thyroid follicular tumors.

Preoperative characterization of thyroid follicular lesions is challenging. Fine-needle aspiration specimens cannot differentiate follicular carcinomas from benign follicular neoplasias. Recently, promising markers have been detected using modern molecular techniques. We conducted a retrospective study to confirm the usefulness of immunohistochemical staining for the protein markers, DDIT3, STT3A (ITM1), ARG2 and FAM129A (C1orf24) in separating benign and malignant thyroid follicular lesions. Formalin-fixed, paraffin-embedded thyroid tissue from 30 in-house cases (15 follicular carcinomas and 15 follicular adenomas), as well as 8 follicular carcinomas and 21 follicular adenomas on tissue microarray slides were stained immunohistochemically for DDIT3, STT3A, ARG2 and FAM129A expression. Control tissue consisted of thyroid parenchyma adjacent to the tumors and 11 separate cases of normal thyroid parenchyma. All in-house cases of follicular adenomas, follicular carcinomas and adjacent normal thyroid tissue showed positive immunostaining with anti-DDIT3 and anti-STT3A. Anti-ARG2 and anti-FAM129A polyclonal antibodies showed positive staining in 20 and 60% of in-house follicular adenomas, and 40 and 87% of in-house follicular carcinomas, respectively. Monoclonal anti-FAM129A demonstrated positive staining in 13 and 33% of in-house follicular adenomas and follicular carcinomas, respectively. Polyclonal anti-DDIT3, -STT3A and -FAM129A antibodies showed positive staining in all tissue microarray slides of follicular carcinoma and in 76, 85 and 81% of the follicular adenomas, respectively. Monoclonal anti-STT3A stained 81% of the follicular adenoma cores. Anti-ARG2 stained positive in 13% of follicular carcinomas and 10% of follicular adenomas on the tissue microarray slides. In conclusion, DDIT3, STT3A, ARG2 and FAM129A immunohistochemistry does not appear to be useful in the diagnosis of thyroid follicular neoplasias, as they do not reliably distinguish follicular thyroid carcinoma from follicular thyroid adenoma.

New oligosaccharyltransferase assay method.

We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

MS analysis of chondroitin polymerization: effects of Mn2+ ions on the stability of UDP-sugars and chondroitin synthesis.

Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn(2+) ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn(2+) ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.

Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

New and simple plate test for screening relative transfructosylation activity of fungi

Several microorganisms are reported to have transfructosylation activity due to fructosyltransferase and/or fructofuranosidase activities. However, the search for other fungi with higher transfructosylation activity remains a challenge. So, a presumptive and indirect colorimetric plate assay for the evaluation of transfructosylation activity in fungi was developed which involved the simultaneous determination in the same plate of glucose and fructose released from sucrose. The method entailed the (a) glucose oxidase-peroxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and (b) fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. The presence of enzymes with transfructosylation activity was identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the fungal colony. In conclusion, the results showed that the method is suitable for screening a large number of fungi due to its simplicity, reproducibility and rapidity and also gives a relative quantitative idea of the transfructosylation activity of different fungi species(AU)

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611.

Different concentrations of sucrose (3-25% w/v) and peptone (2-5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5-17.5% w/v total sugar) and yeast powder (1.5-5% w/v) were used as alternative nutrients for both strains' cultivation. These media were formulated for analysis of cellular growth, beta-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U ( t )) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.

Diagnosis of suspicious thyroid nodules using four protein biomarkers.

PURPOSE: Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples. EXPERIMENTAL DESIGN: Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage-inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions. RESULTS: C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers. CONCLUSIONS: We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.

New and simple plate test for screening relative transfructosylation activity of fungi.

Several microorganisms are reported to have transfructosylation activity due to fructosyltransferase and/or fructofuranosidase activities. However, the search for other fungi with higher transfructosylation activity remains a challenge. So, a presumptive and indirect colorimetric plate assay for the evaluation of transfructosylation activity in fungi was developed which involved the simultaneous determination in the same plate of glucose and fructose released from sucrose. The method entailed the (a) glucose oxidase-peroxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and (b) fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. The presence of enzymes with transfructosylation activity was identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the fungal colony. In conclusion, the results showed that the method is suitable for screening a large number of fungi due to its simplicity, reproducibility and rapidity and also gives a relative quantitative idea of the transfructosylation activity of different fungi species.

A mini exon in the sucrose:sucrose 1-fructosyltransferase gene of wheat.

We previously reported the cloning of a wheat sucrose:sucrose 1-fructosyltransferase (1-SST) cDNA, designated wft2. Wft2 proteins have fructosyltransferase enzyme activity and initiate fructan synthesis (Biosci. Biotechnol. Biochem. 66 (2002) 2297). In the current study, we cloned a genomic DNA fragment carrying the full-length 1-SST gene from winter wheat (Triticum aestivum). The genomic 1-SST gene is 3326 bp in length and contains four exons and three introns. Exon 2 has only 9 bp. This sequence encodes a part of a beta-fructosidase motif (NDPNG), a highly conserved motif found in plant invertases. This is the first report of a mini exon, one of the smallest exons known in plants, being found in a genomic 1-SST gene.

[Methicillin resistance detection in Staphylococcus aureus: comparison between conventional methods and MRSA-Screen latex agglutination technique]. / Detección de meticilino-resistencia en Staphylococcus aureus: comparación de métodos convencionales y aglutinación con MRSA-Screen latex.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the "gold standard" for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100%, agar dilution 97 and 95%, oxacillin agar screen test 100 and 100%, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Evaluation of penicillin binding protein 2a latex agglutination assay for identification of methicillin-resistant Staphylococcus aureus directly from blood cultures.

The penicillin binding protein 2a (PBP2a) latex agglutination test using a blood culture pellet was compared to the oxacillin screen agar method using isolated colonies. For blood cultures positive for Staphylococcus aureus (n = 70), the direct PBP2a test was 18% sensitive and 100% specific. The PBP2a test shows poor sensitivity when used directly with positive blood cultures.

Detección de meticilino-resistencia en Staphylococcus aureus: Comparación de métodos convencionales y aglutinación con mrsa-Screen latex / Methicillin resistance detection in Staphylococcus aureus: Comparison between conventional methods and Mrsa-Screen latex agglutination technique

Staphylococcus aureus meticilino-resistente (MRSA) es un patógeno que ha emergido en las últimas cuatro décadas causando tanto infecciones nosocomiales como de la comunidad. La rápida y precisa detección de MRSA es relevante para guiar una apropiada terapia antibiótica y evitar la diseminación nosocomial de MRSA.En este trabajo se evaluó la eficiencia de métodos convencionales para la detección de meticilino-resistencia como difusión por discos, CIM en medio sólido, screening de oxacilina, y el nuevo test de aglutinación MRSA-Screen latex sobre 100 aislamientos de S. aureus, 79 mecA positivos y 21 mecA negativos. El test de aglutinación MRSA-Screen latex (Denka Seiken, Niigata, Japón) detecta la presencia de la PLP-2a, producto del gen mecA en cepas de S. aureus. La detección del gen mecA por PCR se utilizó como gold standard para comparar los resultados de los diferentes métodos. La sensibilidad y especificidad fueron 97 y 100 % para el método de difusión, 97 y 95 % para la CIM en medio sólido, 100 y 100 % para el screening de oxacilina y 100 y 100 % para MRSA-Screen latex. Todos los métodos presentaron alta sensibilidad y especificidad, pero el “MRSA-Screen latex” mostró la ventaja de poder brindar un resultado confiable, equivalente a la PCR, en sólo 15 minutos.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the “gold standard” for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100 %, agar dilution 97 and 95 %, oxacillin agar screen test 100 and 100 %, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Moenomycin-resistance is associated with vancomycin-intermediate susceptibility in Staphylococcus aureus.

We have previously isolated a vancomycin-intermediate susceptibility mutant from methicillinresistant Staphylococcus aureus(MRSA) strain COL, and demonstrated the increased glycan-chain length and the decreased moenomycin-susceptibility. To further investigate the relationship between the resistance to vancomycin and to moenomycin, we isolated moenomycin-resistant mutants (4-16 fold higher compared to the parent) from 5 MRSA and 2 methicillin-sensitive S. aureus(MSSA) strains. The MRSA mutants showed a decreased susceptibility to vancomycin (2-4 fold), teicoplanin (2-4 fold) and an increased susceptibility to methicillin (2-8 fold). MSSA strains also showed similar results with those of MRSA strains except that there was no alteration of methicillin susceptibility. Among the mutants, three mutants including two MRSA mutants and one MSSA mutant were analyzed by electron microscopy, and they showed thickened cell walls compared to those of the parents. The glycan-chain length of the peptidoglycan of the mutant was shown to be slightly longer than that of the parent, but the muropeptide profile was very similar. The expression levels of all PBPs were similar to those of the parent. Furthermore, the nucleotide sequences of sgtA, sgtB and pbp2 in the mutant were identical to those of the parent. These results indicate that the moenomycin-resistance is closely associated with vancomycin-intermediate susceptibility in S. aureus.