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Background: Through the recent development of analytical technology, antibiotics quantification in the Japanese Pharmacopoeia (JP) has changed from traditional microbiological assays to physicochemical methods with high specificity and precision. However, for several multicomponent antibiotics without typical UV absorption, potency cannot be directly determined using instrumental methods such as high-performance liquid chromatography; therefore, traditional microbiological assays are still used. Gentamicin sulfate (GmS), which consists of three major components, C1, C1a, and C2, is such a typical antibiotic, and its antimicrobial potency continues to be assayed using microbiological methods in JP monographs. Introduction of a physicochemical assay for GmS is needed to help ensure its quality and quantity., Objective: This study aimed to develop quality control measures for GmS that could be complementary to quantitative assays and purity tests specified in the JP., Methods: For each gentamicin C component (C1, C2, and C1a), theoretical potencies were determined based on the quantitative relationship between purity and potency, as measured by quantitative 1H NMR and microbiological assays, respectively. Two lots of the JP reference standard (RS) were used as test samples, with the contents of each component and impurity (sisomicin and garamine) being determined using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)., Results: The ratios of theoretical potency for C1, C2, and C1a were 1.00, 1.21, and 1.80, respectively. The potencies of the GmS JP RSs, which were estimated based on the contents and theoretical potency of each C component, corresponded well with those determined through microbiological assays. Marked differences in impurities (%) between the two RS lots were highlighted by quantifying sisomicin and garamine., Conclusions: The developed analytical procedure enabled the characterization of two different JP RSs in terms of content ratio, potencies, and impurities., Highlights: Novel analytical procedures useful for routine quality control of GmS were developed using HILIC-MS/MS., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In vertebrates, genomic DNA double-strand breaks (DSBs) are removed by non-homologous end-joining processes: classical non-homologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ); or by homology-dependent processes: gene-conversion (GC) and single-strand annealing (SSA). Surprisingly, these repair pathways are not real alternative options restoring genome integrity with equal efficiency, but show instead striking differences in speed, accuracy and cell-cycle-phase dependence. As a consequence, engagement of one pathway may be associated with processing-risks for the genome absent from another pathway. Characterization of engagement-parameters and their consequences is, therefore, essential for understanding effects on the genome of DSB-inducing agents, such as ionizing-radiation (IR). Here, by addressing pathway selection in G2-phase, we discover regulatory confinements in GC with consequences for SSA- and c-NHEJ-engagement. We show pronounced suppression of GC with increasing DSB-load that is not due to RAD51 availability and which is delimited but not defined by 53BP1 and RAD52. Strikingly, at low DSB-loads, GC repairs ∼50% of DSBs, whereas at high DSB-loads its contribution is undetectable. Notably, with increasing DSB-load and the associated suppression of GC, SSA gains ground, while alt-EJ is suppressed. These observations explain earlier, apparently contradictory results and advance our understanding of logic and mechanisms underpinning the wiring between DSB repair pathways., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The adenylate cyclase toxin (ACT) of Bordetella pertussis internalizes its catalytic domain into target cells. ACT can function as a tool for delivering foreign protein antigen moieties into immune effector cells to induce a cytotoxic T lymphocyte response. In this study, we replaced the catalytic domain of ACT with an enzymatically active protein moiety, the S1 (ADP-ribosyltransferase) subunit of pertussis toxin (PT). The S1 moiety was successfully internalized independent of endocytosis into sheep erythrocytes. The introduced polypeptide exhibited ADP-ribosyltransferase activity in CHO cells and induced clustering typical to PT. The results indicate that ACT can act as a vehicle for not only epitopes but also enzymatically active peptides to mammalian cells., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.