Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein “multisubstrate glycosidase A” (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest K_m with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest k_cat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α₁-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.     

Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation

Methylation-specific PCR (MSP) is frequently used to distinguish methylated alleles in the genome. Sequences that have been incompletely converted during bisulfite treatment are frequently co-amplified during MSP. For accurate MSP, it is important to detect methylated sequences in a background of unconverted DNA with a high level of sensitivity. We report here sensitive techniques, bisulfite conversion-specific MSP (BS-MSP) to accurately evaluate CpG methylation. BS-MSP provides accurate results across a wide spectrum of bisulfite conversion levels. BS-MSP is also confirmed to be a useful technique for the routine analysis of clinical tumor specimens that were paraffin-embedded and microdissected. BS-MSP thus provides the powerful features of ease of use and compatibility with paraffin sections. We recommend that methylation analysis should include a step to eliminate unconverted DNA to avoid overestimation of the DNA methylation level in the samples.     

Estimating the spawning activity of fish species using nuclear and mitochondrial environmental DNA concentrations and their ratios

1. During spawning activity, fish release large amounts of sperm and eggs into the water, which has been assumed to cause an increase in environmental DNA (eDNA) levels and nuclear DNA/mitochondrial DNA ratios. To test whether these assumptions are valid and whether nuclear and mitochondrial eDNA analysis can be used to monitor the spawning activity of freshwater fish, we conducted field eDNA surveys and traditional surveys using common carp (Cyprinus carpio), largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis macrochirus) as model species.
2. Fish spawning periods were estimated based on age, as estimated using the body lengths of juveniles collected in the Miharu reservoir in Fukushima, Japan. The results showed that the main spawning periods of largemouth bass and bluegill sunfish were from April to July and from July to August, respectively.
3. Field eDNA surveys were conducted in the Hebisawagawa front reservoir, which is connected to the Miharu reservoir. From March to August 2019 and 2020, weekly eDNA sampling was conducted at three sites, and daily sampling was conducted at six sites from 23 June to 3 July 2020. The eDNA concentrations of the nuclear internal transcribed spacer 1 (ITS1) and mitochondrial cytochrome B (CytB), as well as the ITS1/CytB ratio, were measured for each of the three fish in each water sample. Water temperature had a statistically significant effect on eDNA concentration, probably reflecting the relationship between water temperature and spawning.
4. We created generalised additive mixed models to estimate spawning activity periods based on weekly eDNA data. The estimated periods of spawning activity for common carp, largemouth bass and bluegill sunfish were March to May, May to July, and May to August, respectively. The estimated spawning periods coincided with known fish ecology or the results of traditional methods. This method also has been applied to daily eDNA samples, showing the feasibility of high-resolution estimation of spawning activity.
5. For common carp and bluegill sunfish, we were able to estimate the spawning period using this method. Although the method is affected by biomass and the diffusion and degradation of eDNA, it has the potential to accurately estimating spawning activities. These then can be estimated without conducting laborious traditional surveys, facilitating the monitoring of reproduction by rare, invasive or important fishery species. Further research on the diffusion distance and degradation time of the eDNA concentration peak caused by fish spawning activity may improve the accuracy of monitoring.

     

Amino acid sequence of a novel heat-stable enterotoxin produced by a yst gene-negative strain of Yersinia enterocolitica

Summary A novel heat-stable enterotoxin (designated Y-STb) was isolated and purified to homogeneity from the culture supernatant of a pathogenic but yst gene-negative strain of Yersinia enterocolitica. The amino acid sequence of the toxin was determined to be Lys-Ala-Cys-Asp-Thr-Gln-Thr-Pro-Ser-Pro-Ser-Glu-Glu-Asn-Asp-Trp-Cys-Cys-Glu- Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys. Y-STb was 20-fold more potent (minimum effective dose in the suckling mouse assay was 0.35 pmol) than the previously documented heat-stable enterotoxin (Y-STa) which is produced by yst gene-positive strains of Y. enterocolitica and has a minimum effective dose of 7.8 pmol. The sequence of Y-STb is different from that of Y-STa in the N-terminal half (1–17), but quite similar in the C-terminal half (18–30). To elucidate the effect of 13 amino acid substitutions in Y-STb on enhancing the toxicity, several short analogs of Y-STb were synthesized and their toxicities were compared in the suckling mouse assay. The enhanced enterotoxicity could be ascribed to the addition of a tryptophan residue at the N-terminus of the ST toxic domain which is the minimum structure essential for toxic activity; the presence of an aspartic acid residue at the same position caused a decrease in toxicity.     

Role of cyclophilin B in activation of interferon regulatory factor-3

IRF-3 is a member of the interferon regulatory factors (IRFs) and plays a principal role in the induction of interferon-beta (IFN-beta) by virus infection. Virus infection results in the phosphorylation of IRF-3 by IkappaB kinase epsilon and TANK-binding kinase 1, leading to its dimerization and association with the coactivators CREB-binding protein/p300. The IRF-3 holocomplex translocates to the nucleus, where it induces IFN-beta. In the present study, we examined the molecular mechanism of IRF-3 activation. Using bacterial two-hybrid screening, we isolated molecules that interact with IRF-3. One of these was cyclophilin B, a member of the immunophilins with a cis-trans peptidyl-prolyl isomerase activity. A GST pull-down assay suggested that one of the autoinhibition domains of IRF-3 and the peptidyl-prolyl isomerase domain of cyclophilin B are required for the binding. A knockdown of cyclophilin B expression by RNA interference resulted in the suppression of virus-induced IRF-3 phosphorylation, leading to the inhibition of the subsequent dimerization, association with CREB-binding protein, binding to the target DNA element, and induction of IFN-beta. These findings indicate that cyclophilin B plays a critical role in IRF-3 activation.     

The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway

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Macromolecular ionophores. 1. Chiral recognition properties of poly[(1→6)-2,5-anhydro-D-glucitol] toward racemic amino acid ester

The chiral recognition property of poly[(1→6)-2,5-anhydro-3,4-di-O-alkyl-D-glucitol] ( 1 ) toward racemic RCH (CO₂CH₃)NH₃⁺ · PF₆^? ( 2 · HPF₆) has been studied using a transport system involving an aqueous source and receiving phases separated by a chloroform phase containing 1 . Transport rates for aromatic guests 2a (R = Ph) and 2b (R = CH₂Ph) were faster than those for aliphatic guests, 2c (R = CH(CH₃)₂) and 2d (R = CH₂CH(CH₃)₂), using the polymer substituted with methyl groups ( 1a ). The enantiomeric excess (e.e.) was 10.9% for 2a as a maximum value and decreased in the order of 2a > 2c > 2b = 2d . When the transport of 2a · HPF₆ was carried out using the polymers with 3,4-di-O-methyl ( 1a ), ethyl ( 1b ), allyl ( 1c ), and pentyl ( 1d ) groups, the e.e. was 22.0% for 1d as a maximum value and increased in the order of 1a < 1b < 1c < 1d . The formation of a complex between 1a and 2a · HPF₆ was confirmed by ¹H and ¹³C NMR spectral measurements. © 1995 Wiley-Liss, Inc.     

Failure to Degrade CAT-Tailed Proteins Disrupts Neuronal Morphogenesis and Cell Survival

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Lipase-catalyzed esterification of 2-(4-substituted phenoxy)propionic acids in organic solvents: substituent effect controlling enantioselectivity toward racemic acids

The enantioselectivity for lipase-catalyzed esterifications of 2-(4-substituted phenoxy)propionic acids in organic solvents was found to be mainly controlled by both size (steric) and electronic effects of substituents: H, F, Cl, CF₃ and CH₃. For the similar substituents in size, CF₃ and CH₃, however, their electronic effects play an important role in controlling the enantioselectivity. A model for the enantiorecognition is proposed by the discussion based on the value of the Michaelis constant obtained for the enantiomers.