Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide. A gene coding for the purified beta-N-acetylglucosaminidase was isolated. The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.     

Purification and characterization of chitosanase and Exo-beta-D-glucosaminidase from a Koji mold, Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa exo-beta-D-glucosaminidase,enzyme,named released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

Analysis of control elements for position‐independent expression of human α‐lactalbumin YAC

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high‐expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human α‐lactalbumin gene express the transgene in a position‐independent manner. The 210 kb YAC was thought to have all the elements necessary for position‐independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the α‐lactalbumin gene had position‐independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position‐dependent expression. Therefore, all the elements necessary for position‐independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the α‐lactalbumin gene. Furthermore, we replaced the human α‐lactalbumin promoter with the bovine αS1‐casein promoter in the 210 kb YAC and produced transgenic rats. Position‐dependent expression was observed. The elements required for position‐independent expression of the bovine αS1‐casein gene are different from those required for the human α‐lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity. Mol. Reprod. Dev. 54:17–23, 1999. © 1999 Wiley‐Liss, Inc.     

Targeted delivery to macrophages and dendritic cells by chemically modified mannose ligand-conjugated siRNA

Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.     

Determination of amyloid beta protein precursors harboring active form of proteinase inhibitor domains in cerebrospinal fluid of Alzheimer's disease patients by trypsin-antibody sandwich ELISA

beta-Amyloid protein precursors (APP) having proteinase inhibitor domains (APPI) were quantified by a new sandwich enzyme linked immunosorbent assay for detection of active (free) form of proteinase inhibitors by using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microtiterplate. The concentration of APPs having APPI in cerebrospinal fluid of Alzheimer's disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.     

Properties of the inulinase gene levH1 of Lactobacillus casei IAM 1045; cloning, mutational and biochemical characterization

Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1.     

Possible finger structure in gene A protein of Microviridae

Microvirid phages alpha 3 and phi K encode for A protein which functions in initiation of the viral DNA synthesis. By nucleotide sequencing analysis, we have found that each gene A protein has 'finger motif structure' which conserves two cysteine and histidine residues similar to the consensus sequence deduced from more than thirty finger motifs reported from many eukaryotic regulatory proteins. In closely related phages, phi X174 and G4, we have detected the same structures in their gene A proteins also.     

Characterization of the histidine decarboxylase gene of Staphylococcus epidermidis TYH1 coded on the staphylococcal cassette chromosome

Histamine production from histidine in fermented food results in food spoilage, and is harmful to consumers. From fish-miso, we have isolated a new bacterial strain Staphylococcus epidermidis TYH1, which produced histamine under acidic condition in the medium supplemented with glucose. Using oligonucleotides deduced from the histidine decarboxylase gene (hdcA) of Lactobacillus hilgardii, about 14-kbp DNA region of the TYH1 genome was cloned and sequenced. This region contained two putative genes hdcA(TYH1) and hdcP(TYH1) encoding proteins HdcA(TYH1) (310 amino acid residues) and HdcP(TYH1) (495 residues), respectively. Nucleotide sequence around this hdc cluster showed similarity to SCCpbp4 region of S. epidermidis ATCC 12228. Downstream of the cluster, ccrA, ccrB (Type II, respectively) and pbp4 were located. The CcrA and CcrB proteins catalyzed excision of the hdc cluster from the TYH1 chromosome, upon introduction into the TYH1 strain via multicopy plasmid. When hdcA(TYH1) was expressed in Staphylococcus warneri M, histamine was extracellularly accumulated in dependence on exogenous histidine. These results indicate that the gene encoding a histidine decarboxylase resides in a movable genetic element, SCC. This new element is designated as SCChdc.