1.The action of Pseudomonas aeruginosa biofilms in intrinsic drug resistance.
Yi XIE ; Wen-xiang JIA ; Wei ZENG ; Wei-qing YANG ; Xi CHENG ; Xue-ru LI ; Lan-lan WANG ; Mei KANG ; Zai-rong ZHANG
Chinese Medical Journal 2005;118(19):1615-1622
BACKGROUNDThere is a growing interest in studying the relationship between intrinsic resistance and biofilms resistance to drugs. However, the relationship still remains unclear in the macroscopic bacterial growth. Our study is to illuminate the change of bacterial drug resistance of gyrA mutant and active efflux pump during the development of Pseudomonas aeruginosa (P. aeruginosa) biofilms.
METHODSThe strains of type II topoisomerase gene mutant (gyrA mutant) and multidrug resistance (MDR) efflux pump were clinical isolates and detected by polymerase chain reaction (PCR). The process of bacterial biofilms development was observed by scanning electron microscope. Triparental mating experiments were performed to transfer report gene of green fluorescent protein (GFP) into P. aeruginosa biofilms strains and followed by analysis of bacterial survival rate between intrinsic resistance and biofilms resistance.
RESULTSThe fluorescent strains with pGFPuv could develop mature biofilms on Teflon surface. Before a period of 72 hours, the survival rate of biofilms bacteria and intrinsic resistance strains in ciprofloxacin solution was significantly different (P < 0.05). The survival number of intrinsic resistance strains (gyrA mutation and active efflux pump) was illustriously higher than biofilm strain in the initial stage of biofilms development. After 72 hours incubation, there was no clearly difference between mutants and biofilms strains in the survival rate (P > 0.05). The carbonyl cyanide m-chlorophenylhydrazone and azithromycin could significantly reduce the drug resistance of biofilm strains and efflux pump strains.
CONCLUSIONSIn the development of P. aeruginosa biofilms, the strains of gyrA mutation and MDR efflux could be conferred with new level of drug resistance. When co-cultured mutated strains with biofilm strains, biofilms may play a major role in bacterial resistance. But after 72 hours incubation (a mature biofilms had been developed), there was no clearly difference between the number of mutant strains and biofilm strains.
Biofilms ; drug effects ; Carbonyl Cyanide m-Chlorophenyl Hydrazone ; pharmacology ; Ciprofloxacin ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; Mutation ; Pseudomonas aeruginosa ; drug effects ; genetics
2.Treadmill exercise alleviates neuropathic pain by regulating mitophagy of the anterior cingulate cortex in rats.
Cui LI ; Xiao-Ge WANG ; Shuai YANG ; Yi-Hang LYU ; Xiao-Juan GAO ; Jing CAO ; Wei-Dong ZANG
Acta Physiologica Sinica 2023;75(2):160-170
This study aimed to investigate the effect of treadmill exercise on neuropathic pain and to determine whether mitophagy of the anterior cingulate cortex (ACC) contributes to exercise-mediated amelioration of neuropathic pain. Chronic constriction injury of the sciatic nerve (CCI) was used to establish a neuropathic pain model in Sprague-Dawley (SD) rats. Von-Frey filaments were used to assess the mechanical paw withdrawal threshold (PWT), and a thermal radiation meter was used to assess the thermal paw withdrawal latency (PWL) in rats. qPCR was used to evaluate the mRNA levels of Pink1, Parkin, Fundc1, and Bnip3. Western blot was used to evaluate the protein levels of PINK1 and PARKIN. To determine the impact of the mitophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP) on pain behaviors in CCI rats, 24 SD rats were randomly divided into CCI drug control group (CCI+Veh group), CCI+CCCP low-dose group (CCI+CCCP0.25), CCI+CCCP medium-dose group (CCI+CCCP2.5), and CCI+CCCP high-dose group (CCI+CCCP5). Pain behaviors were assessed on 0, 1, 3, 5, and 7 days after modeling. To explore whether exercise regulates pain through mitophagy, 24 SD rats were divided into sham, CCI, and CCI+Exercise (CCI+Exe) groups. The rats in the CCI+Exe group underwent 4-week low-moderate treadmill training one week after modeling. The mechanical pain and thermal pain behaviors of the rats in each group were assessed on 0, 7, 14, 21, and 35 days after modeling. Western blot was used to detect the levels of the mitophagy-related proteins PINK1, PARKIN, LC3 II/LC3 I, and P62 in ACC tissues. Transmission electron microscopy was used to observe the ultrastructure of mitochondrial morphology in the ACC. The results showed that: (1) Compared with the sham group, the pain thresholds of the ipsilateral side of the CCI group decreased significantly (P < 0.001). Meanwhile, the mRNA and protein levels of Pink1 were significantly higher, and those of Parkin were lower in the CCI group (P < 0.05). (2) Compared with the CCI+Veh group, each CCCP-dose group showed higher mechanical and thermal pain thresholds, and the levels of PINK1 and LC3 II/LC3 I were elevated significantly (P < 0.05, P < 0.01). (3) The pain thresholds of the CCI+Exe group increased significantly compared with those of the CCI group after treadmill intervention (P < 0.001, P < 0.01). Compared with the CCI group, the protein levels of PINK1 and P62 were decreased (P < 0.001, P < 0.01), and the protein levels of PARKIN and LC3 II/LC3 I were increased in the CCI+Exe group (P < 0.01, P < 0.05). Rod-shaped mitochondria were observed in the ACC of CCI+Exe group, and there were little mitochondrial fragmentation, swelling, or vacuoles. The results suggest that the mitochondrial PINK1/PARKIN autophagy pathway is blocked in the ACC of neuropathic pain model rats. Treadmill exercise could restore mitochondrial homeostasis and relieve neuropathic pain via the PINK1/PARKIN pathway.
Rats
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Animals
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Mitophagy/physiology*
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Rats, Sprague-Dawley
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Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology*
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Gyrus Cinguli
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Neuralgia
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Ubiquitin-Protein Ligases/metabolism*
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Protein Kinases
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Membrane Proteins/metabolism*
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Mitochondrial Proteins/metabolism*
3.Inhibitory effects of reserpine and carbonyl cyanide m-chloro-phenylhydrazone on fluoroquinolone resistance of Acinetobacter baumannii.
Wei-feng SHI ; Jian-ping JIANG ; Ning XU ; Zhi-mi HUANG ; Yu-yue WANG
Chinese Medical Journal 2005;118(4):340-343
Acinetobacter baumannii
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drug effects
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genetics
;
metabolism
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Amino Acid Sequence
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Base Sequence
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Carbonyl Cyanide m-Chlorophenyl Hydrazone
;
pharmacology
;
DNA Gyrase
;
genetics
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DNA Topoisomerase IV
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genetics
;
Drug Resistance, Bacterial
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Fluoroquinolones
;
pharmacology
;
Reserpine
;
pharmacology
4.Effect of Ketamine on Apoptosis by Energy Deprivation in Astroglioma Cells using Flow Cytometry System.
Soo Joo CHOI ; Myung Hee KIM ; Seung Woon LIM ; Mi Sook GWAK
Journal of Korean Medical Science 2005;20(1):113-120
Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astroctye functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/ carbonylcyanide mchlorophenylhydrazone (IAA/CCCP) 1.5 mM/ 20 micrometer or 150 micrometer/2 micrometer for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of keta-mine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.
Anesthetics, Dissociative/*pharmacology
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Annexin A5/pharmacology
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Apoptosis
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Astrocytes/metabolism
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Astrocytoma/*drug therapy/pathology
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Brain/pathology
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Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology
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Cell Line, Tumor
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Cell Size
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Cell Survival
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Central Nervous System/drug effects/pathology
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Enzyme Inhibitors/pharmacology
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Flow Cytometry/*methods
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Humans
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Indicators and Reagents/pharmacology
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Iodoacetates/pharmacology
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Ischemia/pathology
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Ketamine/metabolism/*pharmacology
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Light
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Neurons/metabolism/pathology
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Neutrophils/metabolism
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Perfusion
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Propidium/pharmacology
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Scattering, Radiation
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Time Factors
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Uncoupling Agents/pharmacology