儿童呼吸道卡他莫拉菌分离株产β-内酰胺酶情况和耐药性以及菌株BRO酶基因特征

目的研究儿童呼吸道卡他莫拉菌分离株产β-内酰胺酶情况和耐药性以及菌株BRO酶基因特征。方法选择2016年2月-2019年1月因呼吸道感染就诊且从痰液标本中分离得到卡他莫拉菌的患儿,测定抗生素敏感性、产β-内酰胺酶情况、BRO酶基因表达情况。结果产酶菌株对氨苄西林、阿莫西林、头孢克洛、头孢呋辛、头孢吡肟、左氧氟沙星、克拉霉素产生耐药的发生率高于非产酶菌株,BRO-1基因阳性菌株对氨苄西林、阿莫西林、头孢克洛、头孢呋辛、头孢吡肟、左氧氟沙星、克拉霉素产生耐药的发生率高于BRO-2基因阳性菌株;产酶菌株和非产酶菌株、BRO-1基因阳性和BRO-2基因阳性菌株均对亚胺培南敏感,未发生耐药。结论儿童呼吸道卡他莫拉菌产β-内酰胺酶比例高,且BRO-1基因阳性菌株的耐药性强于BRO-2基因阳性菌株。     

Evaluation of susceptibility patterns and BRO β-lactamase types among clinical isolates of Moraxella catarrhalis

The aims of this study were to detect BRO beta-lactamase types and to evaluate any correlation with the susceptibility patterns of 90 clinical isolates of Moraxella catarrhalis. The overall prevalences of the bro-1 and bro-2 genes were 78% and 12%, respectively. Penicillin G MICs for BRO-1+ isolates were significantly higher than those for BRO-2+ isolates. All the isolates were susceptible to amoxycillin-clavulanate, levofloxacin and cefixime. Resistance to clarithromycin, tetracycline and trimethoprim-sulphamethoxazole was 1.1%, 2.2% and 1.1%, respectively. One-step, length-based PCR was an efficient method to screen for BRO beta-lactamase genes.     

Detection and quantification of VEGFR-1 in the nuclei of tumor cells by a new flow cytometry-based method

Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA?=?proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p?=?0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.     

The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.     

Benzodiazepines medazepam and midazolam are activators of pregnane X receptor and weak inducers of CYP3A4: Investigation in primary cultures of human hepatocytes and hepatocarcinoma cell lines

Benzodiazepines have wide-spread used in pharmacotherapy for their anxiolytic, myorelaxant, hypnotic, amnesic and anticonvulsive properties. Despite benzodiazepines are used in clinics over 50 years, they have not been surprisingly tested for capability to induce major drug-metabolizing cytochromes P450. In the current study, we have examined the potency of Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Medazepam, Midazolam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam to induce CYP1A2 and CYP3A4 in primary cultures of human hepatocytes. Benzodiazepines were tested in therapeutic concentrations and in concentrations corresponding to their plasma levels in intoxicated patients. We found weak but significant induction of CYP3A4 mRNA by Midazolam and Medazepam, while other benzodiazepines did not induce CYP3A4 expression. None of the tested compounds induced CYP1A2 mRNA in three independent human hepatocytes cultures. In addition, employing gene reporter assays with transiently transfected hepatocarcinoma cells, we found that tested benzodiazepines did not activate aryl hydrocarbon receptor (AhR), whereas Midazolam and Medazepam slightly activated pregnane X receptor (PXR). Consistently, two-hybrid mammalian assay using hybrid fusion plasmids GAL4-PXR ligand-binding domain (LBD) and VP16-SRC-1-receptor-interacting domain (RID) confirmed PXR activation by Midazolam and Medazepam. In conclusion, Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam can be considered as safe drugs in term of their inability to induce PXR- and AhR-dependent cytochrome P450 enzymes CYP1A2 and CYP3A4. Medazepam and Midazolam slightly activated pregnane X receptor and displayed weak potency to induce CYP3A4 mRNA in human hepatocytes.