Monitoring Quinolone Resistance Due to Mutations in GyrA and ParC in Haemophilus Influenzae（2012-17）

Knowing recent drug-resistant bacteria trends is important for proper antibacterial drug use to improve the prognosis of patients with infectious diseases and for public health. Because multiple quinolone antibacterial agents are simultaneously adopted in hospitals in Japan, we examined whether minimum inhibitory concentrations （MICs） against Haemophilus influenzae differ among quinolones. We determined MICs of six different quinolone antibacterial agents and performed molecular genetic analysis. We investigated β-lactamase-producing and β-lactamase-negative ampicillin-resistant（BLNAR）H. influenzae using the nitrocefin method in parallel. Overall, 144 clinical H. influenzae strains isolated at the Showa University Hospital between 2012 and 2017 were subjected to MIC determination for penicillin/quinolone antibacterial agents using the Clinical and Laboratory Standards Institute broth microdilution method. Amino acid mutations in the quinolone resistance-determining regions were analyzed in the isolates showing an MIC value ≥ 0.25µg/ml of quinolone antibacterial agents. BLNAR isolates increased from 2016 onward. Among quinolone antibacterial agents, all isolates remained susceptible to sitafloxacin. However, for moxifloxacin（MFLX）, strains with an MIC value＝0.5µg/ml were detected every year since 2013 except in 2015. Amino acid mutations were investigated in 17 isolates （11.8％） with MFLX MIC value≥ 0.25µg/ml and confirmed in 11 isolates （7.6％）, of which 9 contained GyrA mutations. The results demonstrated that MFLX was useful for predicting the presence of amino acid mutations and 0.25 was an appropriate MIC threshold for this purpose. This screening procedure may be effective for reducing the inappropriate use of quinolones and controlling the emergence of drug-resistant H. influenzae

Applicability of Licorice Extracts for Treatment of Oral Diseases, Evaluated by Simplified In Vitro Assay Systems with Oral Cells

Licorice extracts contain various useful substances for oral health. Alkaline extract showed potent anti‐HIV activity, whereas flavonoid‐rich water extracts showed potent anti‐HSV activity, closely correlated with polarizability, ionization potential, a number of ring systems, atomic number and mass. Licorice flavonoids showed higher tumor‐specificity against human oral squamous cell carcinoma as compared with human normal oral mesenchymal cells. Glycyrrhiza, at noncytotoxic concentrations, potently inhibited the IL‐1β‐induced inflammation in cultured human gingival and periodontal ligament fibroblasts. Glycyrrhizin, a major component of Glycyrrhiza, showed the highest UV‐protected activity. The results suggest the possible applicability of licorice extracts for several oral diseases and cosmetic products

Analysis of the MYD88 L265P mutation in IgM monoclonal gammopathy by semi-nested polymerase chain reaction-based restriction fragment length polymorphism method

MYD88 L265P mutation causes constitutive activation of NF-κB and possible driver mutation in B-cell lymphoid malignancies. It is frequently detected in Waldenstrom’s macroglobulinemia （WM） （50％-100％）, and its detection is important in diagnostic and therapeutic targets of this syndrome. Standard detection method of MYD88 L265P mutation in clinical practice has yet to be established. We developed semi-nested PCR-based restriction fragment length polymorphism （snPCR-RFLP） to detect the mutation. The snPCR-RFLP method is a modification of the PCR-RFLP method, which uses the restriction enzyme BsiEI that recognizes CGACT/CG, intending to increase detection sensitivity by amplification of mutated allele in the DNA sample using semi-nested PCR before enzyme digestion. The detection sensitivity of snPCR-RFLP was estimated as 0.1％, by detecting mutated allele in wild-type allele in the cloned plasmid DNA, which is comparable with allele-specific （AS） PCR method widely used as sensitive detection method. By analyzing 40 cases with IgM monoclonal gammopathy, snPCR-RFLP detected 29/40 （70％） of all cases, 22/31 （70.9％） of WM, and 6/9 （66.6%） of IgM-type monoclonal gammopathy with undetermined significance （IgMMGUS）, including five cases （three cases of WM and two cases of IgMMGUS） in which the mutation was detected only by snPCR-RFLP but not by Sanger sequencing method. Regarding DNA sample status, particularly five cases, a case was extracted from formalin-fixed paraffin-embedded tissue and four cases were extracted from cells by Ficoll-Hypaque density gradient. In correlation with clinical features, the MYD88 mutation detected by snPCR-RFLP method was associated with the adverse prognostic index （WMIPSS） of WM using patient age, hemoglobin （Hb） level, platelet count, β2MG level, and serum IgM level （p＝0.055）. The snPCR-RFLP method is a clinically useful MYD88 mutation detection method that can be performed in general laboratories

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Accumulation of spermidine/spermine N1-acetyltransferase and alternatively spliced mRNAs as a delayed response of HeLa S3 cells following X-ray irradiation

Purpose : A key enzyme of polyamine catabolism, spermidine / spermine N1-acetyltransferase (SSAT), is responsive to antiproliferative agents. The role of SSAT in cellular responses to X-ray irradiation was examined. Materials and methods : Exponentially growing HeLa S3 cells were irradiated by X-rays, and mRNA levels for SSAT were measured as a function of post-irradiation time through Northern hybridization. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect alternatively spliced SSAT mRNAs. The intracellular polyamine content was measured by the o-phthalaldehyde method and the enzymatic activity of SSAT by the increased amount of acetylated spermidine after incubation.Results: Not only SSAT mRNA, but also an alternatively spliced mRNA accumulated at the initial stage of growth inhibition after the first or second replication of irradiated cells. The maximum fold increase relative to the level of non-irradiated cells was 3.0-3.5 for both transcripts after 5-Gy irradiation. On the other hand, the mRNA of ornithine decarboxylase, a key enzyme of polyamine synthesis, was little influenced by X-ray treatment. Enzymatic activity of SSAT and the acetylspermidine level were elevated after X-ray irradiation. Conclusions : Activation of SSAT and the induction of alternatively spliced mRNA of the SSAT gene play an important role in regulating growth inhibition and cell death after X-ray irradiation

The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif.

The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1(18CRRL) or Cy2(154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the asoociation of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 as to allow cyclin A association

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor,p21

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system,as well as by transcriptional regulation.Generally,ubiquitination is regulated by the phosphorylation of the substrate.In this study,we identified the region of p21 responsible for the regulation of ubiquitination.Since the phosphorylation sites of p21 are distributed in the C-terminal region,we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells.The ubiquitination was observed in the 1-164 (full length)and 1-157 fragments with the same efficiency,but not in the 1-147fragment.The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154,since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21.Furthermore,the 148-157 deleted form of p21 was not ubiquitinated,just like the 1-147 fragment.Thus,the C-terminal 148-157 region,not a ubiquitination site by itself,should contain an essential regulatory region for the efficient ubiquitination of p21