Analysis of oligomeric fermentation substrates and products by high pressure size exclusion chromatography

Summary Commercially available maltodextrins were subjected to high pressure size exclusion chromatography (HPSEC) on Toyo Soda G 2000 PW columns with water as the mobile phase. The elution profiles of these samples will allow researchers to select the correct maltodextrin for growth studies requiring specific dextrin oligomers. Characterization of the chromatography system with standards of known molecular weight allows estimation of the weight average molecular weight of polydisperse dextrins. The enzymatic hydrolysis of dextrin by bacterial-amylase and fungal glucoamylase was also monitored by HPSEC.     

Determination of anti-Aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha

The dynamic growth rate of a single hypha of Aspergillus niger was analysed using an automatic system. A colony of A. niger was in contact with saline, saline containing an antifungal agent, and flushing saline, in sequence. The growth rate of a test hypha selected arbitrarily from the colony responded dynamically to the antifungal agent. The minimum concentration that caused the complete inhibition of hyphal growth was defined as the minimum inhibitory concentration (MIC). The MIC values obtained were compared with those determined by conventional methods based on increasing rate of colony diameter or dry matter weight.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production

L-arabinose isomerase (EC 5.3.1.4) mediates the isomerization of D-galactose into D-tagatose as well as the conversion of L-arabinose into L-ribulose. To investigate the properties of L-arabinose isomerase as a biocatalyst for the conversion of galactose to tagatose, the L-arabinose isomerase of Escherichia coli was characterized. The substrate specificity for L-arabinose was 166-fold higher than that for D-galactose. The optimal pH and temperature for the galactose isomerization reaction were 8.0 and 30 °C, respectively. The enzyme activity was stable for 1 h at temperatures below 35 °C and within a pH range of 8–10. The Michaelis constant, K _m, for galactose was 1480 mM, which is 25-fold higher than that for arabinose. The addition of Fe²⁺ and Mn²⁺ ions enhanced the conversion of galactose to tagatose, whereas the addition of Cu²⁺, Zn²⁺, Hg²⁺, and Fe³⁺ ions inhibited the reaction completely. In the presence of 1 mM Fe²⁺ ions, the K _m for galactose was found to be 300 mM.     

Precise recapitulation of methylation change in early cloned embryos

Change of DNA methylation during preimplantation development is very dynamic, which brings this term to the most attractive experimental target for measuring the capability of cloned embryo to reprogram its somatic genome. However, one weak point is that the preimplantation stage carries little information on genomic sequences showing a site-specific re-methylation after global demethylation; these sequences, if any, may serve as an advanced subject to test how exactly the reprogramming/programming process is recapitulated in early cloned embryos. Here, we report a unique DNA methylation change occurring at bovine neuropeptide galanin gene sequence. The galanin gene sequence in early bovine embryos derived by in vitro fertilization (IVF) maintained a undermethylated status till the morula stage. By the blastocyst, certain CpG sites became methylated specifically, which may be an epigenetic sign for the galanin gene to start a differentiation programme. The same sequence was moderately methylated in somatic donor cell and, after transplanted into an enucleated oocyte by nuclear transfer (NT), came rapidly demethylated to a completion, and then, at the blastocyst stage, re-methylated at exactly the same CpG sites, as observed so in normal blastocysts. The precise recapitulation of normal methylation reprogramming and programming at the galanin gene sequence in bovine cloned embryos gives a cue for the potential of cloned embryo to superintend the epigenetic states of foreign genome, even after global demethylation.     

Thionine increases electricity generation from microbial fuel cell using Saccharomyces cerevisiae and exoelectrogenic mixed culture

Microbial fuel cells (MFCs) have been shown to be capable of clean energy production through the oxidation of biodegradable organic waste using various bacterial species as biocatalysts. In this study we found Saccharomyces cerevisiae, previously known electrochemcially inactive or less active species, can be acclimated with an electron mediator thionine for electrogenic biofilm formation in MFC, and electricity production is improved with facilitation of electron transfer. Power generation of MFC was also significantly increased by thionine with both aerated and non-aerated cathode. With electrochemically active biofilm enriched with swine wastewater, MFC power increased more significantly by addition of thionine. The optimum mediator concentration was 500 mM of thionine with S. cerevisae in MFC with the maximum voltage and current generation in the microbial fuel cell were 420 mV and 700 mA/m(2), respectively. Cyclic voltametry shows that thionine improves oxidizing and reducing capability in both pure culture and acclimated biofilm as compared to non-mediated cell. The results obtained indicated that thionine has great potential to enhance power generation from unmediated yeast or electrochemically active biofilm in MFC.     

A lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform for identification of multiple liver cancer biomarkers in human plasma

Aberrantly glycosylated proteins related to liver cancer progression were captured with specific lectin and identified from human plasma by multiple reaction monitoring (MRM) mass spectrometry as multiple biomarkers for hepatocellular carcinoma (HCC). The lectin fractionation for fucosylated protein glycoforms in human plasma was conducted with a fucose-specific aleuria aurantia lectin (AAL). Following tryptic digestion of the lectin-captured fraction, plasma samples from 30 control cases (including 10 healthy, 10 hepatitis B virus [HBV], and 10 cirrhosis cases) and 10 HCC cases were quantitatively analyzed by MRM to identify which glycoproteins are viable HCC biomarkers. A1AG1, AACT, A1AT, and CERU were found to be potent biomarkers to differentiate HCC plasma from control plasmas. The AUROC generated independently from these four biomarker candidates ranged from 0.73 to 0.92. However, the lectin-coupled MRM assay with multiple combinations of biomarker candidates is superior statistically to those generated from the individual candidates with AUROC more than 0.95, which can be an alternative to the immunoassay inevitably requiring tedious development of multiple antibodies against biomarker candidates to be verified. Eventually the lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform was found to be efficient to identify multiple biomarkers from human plasma according to cancer progression.     

Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.