3株耐碳青霉烯鲍曼不动杆菌临床株耐药机制的研究

目的 研究鲍曼不动杆菌（Acinetobacter baumannii）对碳青霉烯耐药的分子机制。方法 采用琼脂纸片扩散法（KB法）及微量肉汤法筛选出3株对碳青霉烯耐药的鲍曼不动杆菌临床株，PCR扩增blaox-23基因和测序、提取外膜蛋白及进行聚丙烯酰胺凝胶电泳（SDS-PAGE）。结果 3株耐碳青霉烯的鲍曼不动杆菌具有多重耐药性；耐药基因PCR扩增和测序证实3株耐药菌均有blaOX-23 基因；其中1株耐药菌AB173的外膜蛋白电泳条带在28．8ku处出现明显缺失，其余2株耐药菌在该条带处与敏感菌相比较无明显差异。结论 本次试验中对碳青霉烯耐药的鲍曼不动杆菌全部含有blaoxhm基因，其中一株耐药菌还出现了外膜蛋白孔道的缺失可能与其多重耐药性有关。     

耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变研究

目的　研究临床分离的耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变情况。方法　测定临床分离的 5 5株铜绿假单胞菌MIC值 ,从中筛选出 1株敏感菌和 8株耐药菌 ,以标准敏感菌株ATCC2 785 3作为质控菌株。用聚合酶链反应 (PCR)扩增gyrA及parC基因的喹诺酮耐药决定区 (QR DR) ,扩增产物片段长度分别为 35 1bp、397bp。用限制性内切酶SacⅡ消化gyrAPCR产物 ,同时对上述 10株菌的gyrA及parC基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果　有 8株耐菌株的gyrA基因在 83位 (ACC→ATC)有突变 ,导致氨基酸Thr→Ile的改变 ;有 3株高度耐药菌gyrA基因同时在 87位 (GAC→GGC)有突变 ,导致氨基酸Asp→Gly的改变 ;有 4株耐药菌株的parC基因在 87位有TCG→TTG突变 ,导致氨基酸由Ser→Leu的改变。同时具gy rA和parC突变MIC值是仅具gyrA突变菌株MIC值的 2～ 16倍。未发现parC突变单独存在。另外 ,有 6株耐药菌gyrA的 132位有CAC→CAT的突变 ;所有耐药菌株parC基因 115位有GCT→GCG的突变 ,该突变未引起氨基酸的改变。结论　gyrA83、87位突变及parC基因 87位突变都可引起铜绿假单胞菌对氟喹诺酮类药物产生耐药 ,但以gyrA基因 83位突变为主 ,合并gyrA基因 87位及parC基因 87位突变可增加耐药程度。     

临床分离铜绿假单胞菌喹诺酮耐药株gyrA基因突变及其对β-内酰胺类药物敏感性研究

利用PCR、限制性片段多态性分析（RFLP）、DNA单链构像多态性分析（SSCP）和DNA测序等方法，对10株成都地区临床分离耐喹诺酮类药物铜绿假单孢菌gyrA基因进行研究。结果表明，成都地区耐喹诺酮类药物铜绿假单胞菌gyrA的喹诺酮耐药决定区编码等83位氨基酸密码子表现出高频单点突变（10株中有8株），其突变方式为ACC→ATC。gyrA的PCR扩增产物SacII酶切片段与测序结果一致。SSCP的带谱与测序结果比较，除1株（PSA2）的SSCP带谱与标准株相同，但测序结果有点突变外，其余菌株与测序结果一致。因此，利用PCR－SSCP－RFLP系统，可快速、准确的检测耐喹诺酮类药物的铜绿假单胞菌gyrA中至少一个碱基的差异。用二倍稀释法测定喹诺酮和β－内酰胺类药物对耐药突变株体外抗菌活性，结果表明，铜绿假单胞菌gyrA基因突变株对诺氟沙星、环丙沙星、左氧氟沙星、头孢他啶、亚胺培南和哌拉西林表现出不同的敏感性。试验所用8株突变株对诺氟沙星和环丙沙星表现出高水平抗性；3株对左氧氟沙星敏感；3株对头孢他啶和哌拉西林表现出耐药，2株对亚胺培南耐药。     

大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮耐药的相关性

目的:研究大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮类耐药的相关性。方法:采用微量稀释法进行常规药敏试验,筛选3株萘啶酸敏感大肠埃希菌和37株萘啶酸耐药大肠埃希菌株;PCR扩增大肠埃希菌喹诺酮耐药决定区(QRDR)相关gyrA、parC基因,进行聚合酶链反应-单链构象多态性(PCR-SSCP)分析,同时PCR扩增marOR基因;在耐药株选取部分菌株对gyrA、parC及marOR基因进行测序,检测其突变情况,其结果与体外药敏试验结果进行比较,研究其相关性。结果:37株耐药株均出现gyrA基因突变,但对环丙沙星低耐株最低抑菌浓度(MIC)=2mg/L只出现gyrA单位点突变,而parC基因未发生突变;环丙沙星高耐株(MIC=64mg/L)gyrA基因出现3个位点突变,parC基因出现单位点突变;在环丙沙星高耐株(MIC=256mg/L),并伴有其他类抗菌药物的多重耐药时,除了出现gyrA和parC基因双位点突变,同时检测到marOR基因的多位点突变。结论:gyrA和parC基因突变在大肠埃希菌对喹诺酮耐药中起着重要作用,gyrA和parC基因突变的程度与大肠埃希菌耐药水平有关,marOR基因多位点突变在多重耐药机制中具有一定的作用。     

耐氟喹诺酮类铜绿假单胞菌的gyrA基因突变研究

目的：研究临床分离的耐氟喹诺酮类药物铜绿假单胞菌gyrA基因突变情况。方法：测定临床分离的55株铜绿假单胞菌的MIC值，从中筛选出1株敏感菌和8株耐药菌。以标准敏感菌株ATCC27853作为质控菌株，用聚合酶链反应（PCR）扩增gyrA基因的喹诺酮耐药决定区（QRDR），扩增产物片段长度为350bp。用限制性内酶SacⅡ消化PCR产物，同时对上述10株菌的gyrA基因的喹诺酮决定区（QRDR）进行PCR－DNA直接测序分析。结果：临床分离铜绿假单胞菌敏感菌株的gyrA基因QRDR经限制性内切酶SacⅡ消化后出现118bp,232bp两条片段，表明该酶切位点未发生突变，此结果经DNA序列析证实。而耐药菌在酶切后均为一条片段，表明该酶切位点消失，经DNA序列分析发现，8株耐药株在83位（ACC→ATC）均有突变，该单位突变引起氨基酸由Thr→Ile的改变；其中有3株高度耐药菌同时发现在87位（GAC→GGC）有突变，该单位点突变引起氨基酸由Asp→Gly的改变，但没有发现87位点突变单独存在，且双位点突变菌株的MIC值与单一位点突变的MIC值相比，有显著的升高，MIC增加4－32倍，除此之外，有6株耐药菌株在132位有一静止突变（CAC→CAT），该 突变未引起氨基酸的改变。结论：gyrA基因突变是铜绿假单胞菌对氟喹酮类药物产生耐药的主要机制之一，铜绿假单胞菌gyrA上83位和87位突变最为常见。     

铜绿假单胞菌环丙沙星耐药株Ⅱ类拓扑异构酶基因突变的研究

用PCR方法，扩增26株临床分离铜绿假单力环丙沙星耐药株、4株环丙沙星敏感菌株和PA01菌株的Ⅱ类拓扑异构酶gyrA、gyrB、parC和parE基因的喹诺酮类药物耐药决定区（QRDR）基因片断；并对铜绿假单胞菌Ⅱ类拓扑异构酶基因突变与耐药性关系进行研究。结果表明90％以上的铜绿假单胞菌耐药株表现出Ⅱ类拓扑异构酶基因突变（26株中有24株）。突变主要发生在gyrA基因（22株）和parC基因（14株）上，其中gyrA基因的第83位氨基酸密码子表现出高频突变（22株中有21株，ACC→ATC，占90％），parC基因第80位氨基酸密码子也表现出较高的突变率（14株中有12株，TCG→TTG，占60％），4株耐药株的gyrB和3株耐药株的parE基因发生单点突变，2株耐药株Ⅱ类拓扑异构酶基因朱检测到突变。用二倍稀释法，测定部分喹诺酮和β→内酰胺类药物对耐药突变株的体外抗菌活性。表明铜绿假单胞菌Ⅱ类拓扑异构酶基因突变株对诺氟沙星、左氧氟沙星、哌拉西林、头孢他啶和亚胺培南表现出不同的敏感性。试验所用24株突变株对诺氟沙星呈现100％的耐药；对左氧氟沙星65％的耐药；40％的菌株对哌拉西林表现出耐药；30％的菌株对头孢他啶耐药；75％的菌株对亚胺培南敏感。     

淋球菌的耐药性分析及其耐氟喹诺酮的分子机制研究

目的 检测108株临床分离淋球菌(NG)对9种药物的敏感性,并检测喹诺酮类耐药淋球菌对环丙沙星的MIC以及gyrA和parC基因突变情况.方法 用KB法检测淋球菌对9种药物的敏感性,用E-test法检测耐药菌株对环丙沙星的MIC,用PCR和DNA测序及序列比较检测基因突变.结果 淋球菌对大观霉素最敏感,其次为头孢曲松、头孢他啶、阿米卡星、四环素、红霉素和青霉素,对喹诺酮类药物高度耐药.环丙沙星的MIC范围在0.008-32μg/ml之间,10株淋球菌喹诺酮类耐药中有6株存在gyrA的Ser91→Phe突变的同时还存在gyrA的Asp95→Gly和/或parC的Aap86/Asn、Ser87→Arg/Ile,有4株只有gyrA基因Ser91→Phe的突变.结论 在淋球菌治疗中可首选大观霉素,其次为头孢曲松和头孢他啶.gyrA和parC基因突变与淋球菌耐喹诺酮类药物密切相关.     

肺炎克雷伯杆菌对氟喹诺酮类抗生素耐药机制的研究

目的研究临床分离的耐氟喹诺酮类药物肺炎克雷伯杆菌的耐药机制。方法临床分离、鉴定的肺炎克雷伯杆菌采用琼脂二倍稀释法测定氟喹诺酮的MIC,从中选取10株耐氟喹诺酮的菌株。通过聚合酶链反应(PCR)扩增gyrA基因和parC基因,扩增产物片段长度分别为6253、19 bp,经纯化后测序。用琼脂二倍稀释的方法测定5种氟喹诺酮类单独使用及与泵抑制剂CCCP合用后的MIC。结果5株耐氟喹诺酮肺炎克雷伯杆菌的gyrA和parC基因经测序分析,显示gyrA的第83位(TCC→ATC/TTG)均出现了突变,引起了相应氨基酸的改变(Ser-83→Ile/Leu)。其中有一株同时也出现了第87位点(GAC→AAC)的改变,氨基酸也由Asp→Asn。parC有4株出现了第80位点突变(AGC→ATC),引起氨基酸由Ser→Ile的改变。MIC测定结果显示,有一株肺炎克雷伯杆菌在单用5种氟喹诺酮类药物及与CCCP合用后,MIC降低了8~32倍;两株肺炎克雷伯杆菌分别对环丙沙星及左氧氟沙星所测的MIC降低了32倍和4倍;还有一株对左氧氟沙星和司帕沙星在使用CCCP前后的MIC降低了4倍。结论gyrA和parC基因突变是肺炎克雷杆菌对氟喹诺酮类产生耐药机制的主要原因,主动外排机制也是肺炎克雷伯杆菌耐氟喹诺酮类抗生素的因素之一。     

铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系研究

目的:研究铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系,并对聚合酶联反应(PCR)-限制性片段长度多态性(RFLP)-DNA单链构象多态性(SSCP)分析铜绿假单胞菌临床分离株gyrA基因突变的可行性进行评估。方法:以铜绿假单胞菌临床分离株gyrA基因序列为靶序列,用PCR、PCR-RFLP、PCR-SSCP、DNA测序等方法对铜绿假单胞菌ATCC27853及16株临床分离株gyrA基因突变进行对比研究。结果:在8株耐环丙沙星铜绿假单胞菌中,有6株gyrA基因的83位表现出单点突变,其突变方式全为ACC→ATC,导致氨基酸苏氨酸→异亮氨酸的改变;gyrA基因的PCR扩增产物SacⅡ酶切片段与测序结果一致;SSCP分析结果显示,16株细菌中仅2株gyrA带型与ATCC27853相同,其它菌株gyrA带型与ATCC27853均不同。结论:临床分离的铜绿假单胞菌对喹诺酮类药物耐药的分子机制主要表现为gyrA基因83位氨基酸密码子突变,应用PCR-RFLP-SSCP系统可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中碱基的变异。     

两种氟喹诺酮类药物对肺炎克雷伯菌防耐药突变浓度及耐药突变体耐药性的研究

目的测定两种氟喹诺酮类药物（FQNs）对肺炎克雷伯菌（KP）的防耐药突变浓度（MPC），比较其防耐药突变能力，了解突变耐药菌对FQNs的耐药性。方法肉汤法富集10^10CFU／ml的菌液接种于不同浓度环丙沙星及加替沙星琼脂平皿上，采用琼脂二倍稀释法测定环丙沙星、加替沙星对临床分离肺炎克雷伯菌、ATCC700603及其耐药突变体的最低抑菌浓度（MIC）、防耐药突变浓度（MPC）。对不同药物浓度筛选出的耐药突变株进行编码拓扑异构酶VIC亚单位基因parC和回旋酶A亚单位基因gyrA喹诺酮耐药决定区的PCR扩增和测序，并测定外排泵抑制剂羟基氰氯苯腙（Carbonyl Cyanide m-chlorophenylhydrazone，CCCP）对环丙沙星和加替沙星MIC的影响。结果环丙沙星、加替沙星对ATCC700603的MPC分别为4、1.6mg／L，细菌耐药选择指数分别为16、4。两种药物的耐药突变体均出现了gyrA的第83位（TCC→ATC／TTG）均出现了突变，引起了相应氨基酸的改变（Ser-83→Ile／Leu）。其中有一株同时也出现了第87位点（GAC→AAC）的改变，氨基酸也由Asp→Asn。除第二步突变体parC第80位点突变（AGC→ATC），引起氨基酸由Ser→Ile的改变外，其余几株均没有发生parC的突变。MIC测定结果显示，单用两种FQNs及合用CCCP的结果一样。结论加替沙星限制KP耐药突变株选择的能力强于环丙沙星，KP的耐药突变株对FQNs耐药的主要原因是gyrA和parC基因突变。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Gender-related symptoms and correlates of alcohol dependence among men and women with a lifetime diagnosis of alcohol use disorders

This study explored gender-related symptoms and correlates of alcohol dependence in a crosssectional study of 150 men and 150 women with a lifetime diagnosis of alcohol use disorders (AUD). Participants were recruited in equal numbers from treatment settings, correctional centres and the general community. Standardized measures were used to determine participants' use of substances, history of psychiatric disorders and psychosocial stress, their sensation seeking and family history of substance use and mental health disorders. Multivariate analyses were used to detect patterns of variables associated with gender and the lifetime severity of AUD. Men had a longer history of severe AUD than women. Women had similar levels of alcohol dependence and medical and psychological sequelae as men, despite 6 fewer years of AUD. More women than men had a history of severe psychosocial stress, severe dependence on other substances and antecedent mental health problems, especially mood and anxiety disorders. There were differences in family history of alcohol-related problems approximating same-gender aggregation. The severity of a lifetime AUD was predicted by its earlier age at onset and the occurrence of other disorders, especially anxiety, among both men and women. The limitations in the generalizability of these findings due to sample idiosyncrasies are discussed.