Structure-Function Analysis of CYP105A1 in the Metabolism of Nonsteroidal Anti-inflammatory Drugs.

CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that CYP105A1 wild type and its variants metabolize 12 types of nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited a high activity against many NSAIDs.

Expression of Recombinant Human α-Glucosidase in HEK293 Cells.

In mammals, intestinal α-glucosidase exists as a maltase-glucoamylase complex (MGAM) and a sucrase-isomaltase complex (SI). In this study, we transiently expressed human MGAM and SI in human embryonic kidney 293 (HEK293) cells. At pH 6.

Unveiling the reaction mechanism of arginine decarboxylase in Aspergillus oryzae: Insights from crystal structure analysis.

Agmatine, a natural polyamine also known as 4-aminobutyl-guanidine, is biosynthesized from arginine by decarboxylation. Aspergillus oryzae contains high amounts of agmatine, suggesting highly active arginine decarboxylase (ADC) in this organism. However, genome analysis revealed no ADC homolog in A.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK).

Use of human Caco-2 cells and HPAE-PAD for α-glucosidase assay.

To measure α-glucosidase activity, rat intestinal acetone powder is commonly used as a source of α-glucosidase, and the mutarotase-glucose oxidase (GOD) methods commonly used to quantitate glucose produced by enzymatic hydrolysis of the substrates. In this study, we compared human Caco-2 cell extracts with rat intestinal acetone powder extracts. We also compared high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with the mutarotase-GOD method.

Adenosine triphosphate induces amorphous aggregation of amyloid β by increasing Aβ dynamics.

Amyloid β (Aβ) aggregates into two distinct fibril and amorphous forms in the brains of patients with Alzheimer's disease. Adenosine triphosphate (ATP) is a biological hydrotrope that causes Aβ to form amorphous aggregates and inhibit fibril formation at physiological concentrations. Based on diffracted X-ray blinking (DXB) analysis, the dynamics of Aβ significantly increased immediately after ATP was added compared to those in the absence and presence of ADP and AMP, and the effect diminished after 30 min as the aggregates formed.

Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR.

XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type XynR (WT) > 15 other variants. In this study, we sought to elucidate the mechanisms underlying the variable activity of these different variants. Crystallographic analysis revealed that the Ca ion near position 315 in WT was absent in the T315Q variant.

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

Background: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility.

Detection of SARS-CoV-2 spike protein D614G mutation using μTGGE.

Background: The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (μTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein.

Methods: The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to μTGGE analysis.

Conservative treatment of traumatic finger amputations using negative-pressure wound therapy.

Replantation is widely regarded as the first choice of treatment for finger amputations. However, if the fingertip of a traumatic finger amputation is missing after an injury, the following procedures are often performed to reconstruct this portion: flap surgery, stump surgery, or conservative treatment, including occlusive dressings. To our knowledge, no existing English literature reports using negative-pressure wound therapy (NPWT) to treat traumatic finger amputations.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells.

Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses.

Grimontia hollisae collagenase (Ghcol) exhibits high collagen-degrading activity. To explore its catalytic mechanism, its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO)-complexed crystal structure was determined at 2.0 Å resolution.

Construction and characterization of ribonuclease H2 C subunit-knockout NIH3T3 cells.

Mammalian ribonuclease (RNase) H2 is a trimer consisting of catalytic A and accessory B and C subunits. RNase H2 is involved in the removal of misincorporated ribonucleotides from genomic DNA. In humans, mutations in RNase H2 gene cause a severe neuroinflammatory disorder, Aicardi-Goutières syndrome (AGS).

Purification and characterization of a serine protease from the fruit of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease (FSP). Here, we purified FSP to homogeneity from the fruit of F. carica cultivar Masui Dauphine.

Analysis of ribonucleotide content in the genomic DNA of ribonuclease H2 A subunit (RH2A)-knockout NIH3T3 cells after transient expression of wild-type RH2A or RH2A variants with an Aicardi-Goutières syndrome-causing mutation.

Ribonuclease (RNase) H2 is involved in the removal of ribonucleotides embedded in genomic DNA. Eukaryotic RNase H2 is a heterotrimer consisting of the catalytic A subunit (RH2A) and the accessory B and C subunits. This study aimed to compare the cellular activities of wild-type ribonuclease (RNase) H2 and its variants with a mutation causing neuroinflammatory autoimmune disease, Aicardi-Goutières syndrome (AGS).

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.

Metabolism of non-steroidal anti-inflammatory drugs (NSAIDs) by Streptomyces griseolus CYP105A1 and its variants.

In the field of drug development, technology for producing human metabolites at a low cost is required. In this study, we explored the possibility of using prokaryotic water-soluble cytochrome P450 (CYP) to produce human metabolites. Streptomyces griseolus CYP105A1 metabolizes various non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, meclofenamic acid, and ibuprofen.

Comparison of the stability of CYP105A1 and its variants engineered for production of active forms of vitamin D.

CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy vitamin D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for vitamin D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including 5 variants (R73A/R84A, M239A, R73A/R84A/M239A (=TriA), TriA/E90A, and TriA/E90D), was examined.

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.

Background: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA.

Methods: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified.

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets.

Inhibitory effect of Morus australis leaf extract and its component iminosugars on intestinal carbohydrate-digesting enzymes.

α-Amylase and α-glucosidase are central enzymes involved in the digestion of carbohydrates. α-Glucosidase includes maltase-glucoamylase and sucrase-isomaltase. We have previously performed the kinetic analysis of the inhibitory effects of powdered or roasted Morus australis leaf extract and its component iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-d-galactopyranosyl deoxynojirimycin (GAL-DNJ) on the activity of maltase.

Preceding direct oral anticoagulant administration reduces the severity of stroke in patients with atrial fibrillation - K-PLUS registry.

Background: Stroke severity can be mitigated by preceding anticoagulant administration in acute ischemic stroke patients with atrial fibrillation (AF). We investigated if such mitigative effects are different between warfarin and direct oral anticoagulants (DOACs).

Material And Methods: We collected data from a regional multicenter stroke registry.