Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Acid carboxypeptidase III from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The optimum activity of the enzyme occurred at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, αα′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 61,000. The enzyme hydrolyzed the peptides that possess masked or bulky N-terminal.     

Polyacrylamide Gel Disc Electrophoresis of Alkaline Proteinases from Aspergillus Species

Ciliatine (2-aminoethylphosphonic acid) (76 mg) was isolated from 72 g of lipids of the oyster with a combination of ion exchange chromatographic techniques and was identified from the results of elementary analysis, infrared spectrum, and chromatographic behaviors. The phosphonic acid was also detected in hydrolysates of a chloroform-methanol insoluble fraction of the oyster. It has been demonstrated that the oyster contains high concentration of ciliatine.     

A New Metabolite, 6-Oxo-7-azatridecanedioic Acid,a Microbial Product from Cyclic Dimer of ε-Caprolactam

A series of partially and heterogeneously N-acylated chitosans was prepared and isolated in 50 ~ 100% yields. The structure of N-acyl groups influenced the gelation. The minimum requirement for the gelation was defined as ca. 0.4 N-lauroyl (C₁₂), ca. 0.6 N-fatty acyl (C₃–C₁₀) or ca. 0.7 N-benzoyl groups per hexosaminide residue. However, the gelation did not occur with N-high fatty acyl (C₁₄-C₁₈) groups.1)     

The Action of Peptidases from Aspergillus sojae on Soybean Proteins

To elucidate the mechanism of light-activation of pyruvate P_L dikinase in maize leaf, the inactive form was purified to homogeneity from dark-treated leaves using an activation system to locate it. The purification procedure included ammonium sulfate-fractionation followed by conventional chromatography.

The homogeneous enzyme after maximal activation had a specific activity comparable to that of the active enzyme obtained from non-dark-treated plants. The enzyme was indistinguishable from the active one in its molecular size and charge and in the amino acid composition of its acid-hydrolysate.     

A Comparative Evaluation of Unsupervised Anomaly Detection Algorithms for Multivariate Data

Anomaly detection is the process of identifying unexpected items or events in datasets, which differ from the norm. In contrast to standard classification tasks, anomaly detection is often applied on unlabeled data, taking only the internal structure of the dataset into account. This challenge is known as unsupervised anomaly detection and is addressed in many practical applications, for example in network intrusion detection, fraud detection as well as in the life science and medical domain. Dozens of algorithms have been proposed in this area, but unfortunately the research community still lacks a comparative universal evaluation as well as common publicly available datasets. These shortcomings are addressed in this study, where 19 different unsupervised anomaly detection algorithms are evaluated on 10 different datasets from multiple application domains. By publishing the source code and the datasets, this paper aims to be a new well-funded basis for unsupervised anomaly detection research. Additionally, this evaluation reveals the strengths and weaknesses of the different approaches for the first time. Besides the anomaly detection performance, computational effort, the impact of parameter settings as well as the global/local anomaly detection behavior is outlined. As a conclusion, we give an advise on algorithm selection for typical real-world tasks.     

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.     

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C_H1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V_H-C_H1 and V_L-C_L was improved by the C_H1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.     

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.