Purification and characterization of endo-beta-N-acetylglucosaminidase of Aspergillus oryzae

An endo-beta-N-acetylglucosaminidase which hydrolyzes the N,N'-diacetylchitobiosyl linkage in asparagine-linked oligosaccharides was purified from the enzyme product of Aspergillus oryzae. Its substrate specificity was similar to that of endo-beta-N-acetylglucosaminidase H from Streptomyces griseus with respect to the relative activities toward the glycopeptides obtained from ovalbumin and bovine IgG. The present endoglycosidase exhibited a broad optimum pH range and was relatively stable. Metal ions, chelating agents and D-mannose did not have a significant effect on the enzyme activity.     

Coexistence of catecholamine and methionine enkephalin-Arg6-Gly7-Leu8 in neurons of the rat ventrolateral medulla oblongata

Summary An overlapping distribution of catecholamine-containing cells and proenkephaline—A derived peptide-containing neurons have been identified in the rat medulla oblongata. However, it is not evident whether the coexistence of these bioactive substances occurs in the same neurons or not. Therefore, we examined the coexistence of catecholamine and methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), a proenkephaline—A derived peptide, using a combination of histofluorescence and peroxidase-anti-peroxidase (PAP) immunohistochemical (modified formaldehyde-glutalaldehyde (Faglu)) methods on the same tissue sections. We found one third of A1/C1 catecholamine fluorescent cells show MEAGL-like immunoreactivity.     

Synthesis of the mating factor of Saccharomyces cerevisiae and its truncated peptides : the structure-activity relationship.

The mating factor of Saccharomyces cerevisiae, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, has been synthesized to confirm the structure of the natural mating factor. The tridecapeptide has the same biological activity as the natural mating factor. From the studies on the biological activity of its truncated peptide synthesized the minimum sequence of the peptide require for the mating factor was deduced to be as His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln.     

Identification of plasma inactive renin as prorenin with a site-directed antibody

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.     

Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus

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Identification of a novel advanced glycation end product derived from lactaldehyde

Advanced glycation end products (AGEs) are implicated in the development of diabetic complications via the receptor for AGEs (RAGE). We have reported that the 3-hydroxypyridinium (3HP)-containing AGEs derived from α-hydroxyaldehydes physically interact with RAGE and show cytotoxicity. Lactaldehyde (LA) is formed from a reaction between threonine and myeloperoxidase, but no LA-derived AGEs have been characterized. Here, we identify the structure and physiological effects of an AGE derived from LA. We isolated a novel 3HP derivative, 2-acetamido-6-(3-hydroxy-5-methyl-pyridin-1-ium-1-yl)hexanoate, named as N-acetyl-LAPL (lactaldehyde-derived pyridinium-type lysine adduct), from a mixture of LA with N^α-acetyl-L-lysine. LAPL was also detected in the LA-modified protein. LAPL elicited toxicity in PC12 neuronal cells, but the effect was suppressed by the soluble form of RAGE as a decoy receptor. Moreover, surface plasmon resonance-based analysis revealed that LAPL specifically binds to recombinant RAGE. These results indicate that LA generates an AGE containing the 3HP moiety and contributes to RAGE-dependent cytotoxicity.

Abbreviations: AGEs: advanced glycation end products; RAGE: receptor for advanced glycation end products; 3HP: 3-hydroxypyridinium; LA: lactaldehyde; LAPL: lactaldehyde-derived pyridinium-type lysine adduct; BSA: bovine serum albumin; GLAP: glyceraldehyde-derived pyridinium; MPO: myeloperoxidase; HFBA: heptafluorobutyric acid; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; LC-ESI-QTOF-MS: liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry; NMR: nuclear magnetic resonance; LA-BSA: lactaldehyde-modified bovine serum albumin; PBS: phosphate buffered saline, GST, glutathione S-transferase; SPR: surface plasmon resonance; OP-lysine: 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate; GLO1: glyoxalase 1; MG, methylglyoxal     

Biomass-dependent emission of environmental DNA in jack mackerel Trachurus japonicus juveniles

Environmental DNA (eDNA) from juvenile jack mackerel Trachurus japonicus was detected in tanks with 1, 3, 10, or 30 individuals per tank. Quantitative PCR using a set of species-specific primers and a probe revealed that the concentration of eDNA increased almost linearly with the density of fish. The coefficient of determination (R²) in the linear regression was lower than values previously reported for freshwater fishes in similar settings.     

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1’s active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.