Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

万古霉素耐药肠球菌基因分型及耐药性分析

目的了解本地区万古霉素耐药肠球菌(Vancomycin resistant Enterococci,VRE)耐药基因型别及耐药性,为临床治疗提供依据。方法用PCR方法检测56株VRE的耐药基因vanA、vanB、vanC、vanD、vanE和vanG;用K-B法检测其对临床常用14种抗菌药物的药敏,并用肉汤稀释法检测万古霉素和替考拉宁的药敏。结果 56株VRE中,vanA阳性的43株;vanB、vanC、vanD、vanE和vanG阳性0株;未检测到万古霉素耐药相关基因的13株。2011-2014年万古霉素和替考拉宁的MIC值呈逐年向左漂移趋势,与同期替考拉宁的使用有关。结论本研究所收集VRE对万古霉素的耐药大部分为高水平耐药,所携带的耐药基因类型主要为vanA,另有其他未知基因型以及少数vanA阳性但是表现为万古霉素敏感菌株。     

In vitro selection of drug resistant Schistosoma mansoni

Schistosomules of Schistosoma mansoni were cultured for 3 days in the presence of schistosomicides and then inoculated intraperitoneally into mice. Drug concentrations killing greater than 99.8% of schistosomules were amoscanate 0.1 p.p.m., oltipraz, 0.5 p.p.m., oxamniquine 240 p.p.m., praziquantel 8 p.p.m. Comparison of drug response of the unselected and selected strains as adult worms in mice showed an increase in tolerance to amoscanate, oltipraz and oxamniquine, but not praziquantel. The oxamniquine tolerant strain did not respond to oxamniquine at 500 mg kg⁻¹. The unselected strain increased in tolerance to three drugs during routine passage in the laboratory. Greater numbers of schistosomules derived from snails exposed to ethyl methane sulfonate appeared to survive culture in metrifonate, suggesting that it may be possible to produce drug resistant schistosomes by mutation and selection.     

Intracellular distribution of anthracyclines in drug resistant cells

The unresponsiveness of multidrug resistant tumor cells to antineoplastic chemotherapy is often associated with reduced cellular drug accumulation accomplished by overexpressed transport molecules. Moreover, intracellular drug distribution in resistant cells appears to be remarkably different when compared to their wild type counterparts. In the present paper, we report observations on the intracellular accumulation and distribution of doxorubicin, an antitumoral agent widely employed in chemotherapy, in sensitive and resistant cultured tumor cells. The inherent fluorescence of doxorubicin allowed us to follow its fate in living cells by laser scanning confocal microscopy. This study included flow cytometric analysis of drug uptake and efflux and analysis of the presence of the well known drug transporter P-glycoprotein. Morphological, immunocytochemical and functional data evidentiated the Golgi apparatus as the preferential intracytoplasmic site of drug accumulation in resistant cells, capable of sequestering doxorubicin away from the nuclear target. Moreover, P-glycoprotein has been found located in the Golgi apparatus in drug induced resistant cells and in intrinsic resistant cells, such as melanoma cells. Thus, this organelle seems to play a pivotal role in the intracellular distribution of doxorubicin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chorology of Liliaceae (S. Str.) and Its Bearing on the Chinese Flora

The geographical distribution of Liliaceae (s. str. ) is analysed on the basis of the floristic regions proposed by Takhtajan. Some conclusions may be proposed as follows: 1.Liliaceae (s. str. ) consists of nine genera and about 513 species, distributed primarily in the north temperate zone. Statistics shows clearly that the Irano-Turanian Region is most abundant in number of species, The Eastern Asian Region with total nine genera of the family is the diversity centre of Liliaceae (s. str. ). 2. The distribution patterns of the genera may be divided into: 1 ) North temperate distribution pattern: Lloydia, Erythronium, Fritillaria and Lilium; 2) Old world temperate distribution pattern: Gagea and Tulipa; 3) West Asia to Himalayas and Southwest China distribution pattern: Notholirion; 4) East Asia distribution pattern: Cardiocrinum and Nomocharis. 3. The Sino-Himalayas is one of the key regions in studying the evolution of Liliaceae (s. str. ) All nine genera occur in the Eastern Asian Region with most species distributed in Southwest and Northwest China. Chorologically, five genera (Fritillaria, Lilium, Cardiocrinum, Nomocharis and Notholirion) of the Lilieae are overlapped each other in the Sino-Himalayas, showing its diversity centre in this region. The Lilieae is a main stock in the Liliaceae (s. str. ), The genus Nomocharis in this tribe may have been newly differentiated from Lilium in the course of the uplift of Qinghai-Xizang Plateau, a view also supported by Xie Xiao-yang et al.. The place of its origin was considered to be in the southern part of the Hengduan Mountains. 4. The distributions of some species in Liliaceae (s. str. ) seem to be significant for dividing some floristic regions: 1 ) Some species of Fritillaria (F. unibracteata Hsiao et K. C. Hsia, F. przewalski Maxim. ex Batal. , F. crassicaulis S. C. Chen, F. cirrhosa D. Don. , F. delavayi Franch. , F. dajinensis S. C. Chen, F. davidii Franch. , F. sinica S. C. Chen and F. sichuanica S. C.Chen) are only distributed in Sino-Himalayan forest subkingdom, while others (F. taiparensis P. Y. Li, F. yuzhongensis S. C. Chen, F. monantha Migo, F. anhuiensis S. C. Chen et S. F. Yin, F. thunbergii Miq. , F. maximowizii Freyn and F. ussuriensis Maxim. ) are restricted to Sino-Japan forest subkingdom. They show a clearly demarcation line between the two subkingdoms, which is identical with what proposed by Wu Cheng-yih. 2) The Eastern Asian Region can be divided into two subkingdoms on the basis of the distribution pattern of the genus Cardiocrinum; C. giganteum (Wall.) Makino and C. gigateum var. yunnanense Leichtlin ex Elwes are restricted to Sino-Himalayan forest subkingdom. C. cathayanum (Wilson) Stearn and C. cordatum (Thunb.) Makino are only found in Sino-Japan forest subkingdom. 3) The distributions of Gagea pauciflora Turcz. , G. triflora(Ledeb.) Roem. et Schult. G. hiensis Pasch, Lloydia tibetica Baker ex Oliver, L. oxycarpa Franch. and L. flavonutans Hara are indicative of a demarcation line between Irano-Turanica Region and Eastern Asian Region. 5. The genus Notholirion occurs in the Eastern Asian Region and Irano-Turanian Region, showing the relationships between the two regions and also between the Chinese flora and Ancient Mediterraneam flora.     

耐亚胺培南黄杆菌的耐药性分析

目的探讨耐亚胺培南黄杆菌的ESBLs和AmpC检出率和耐药率。方法采用ESBLs确认试验检测ESBLs,头孢西丁三维试验检测AmpC,用K-B法进行药敏试验。结果1011株耐亚胺培南黄杆菌中,产ESBLs株787株,占77.8%;检出AmpC 549株,占54.3%;检出同产ESBLs AmpC株439株,占51.6%,其中ESBLs 高产AmpC 288株,占65.6%;ESBLs 诱导AmpC 151株,占34.4%;黄杆菌对β-内酰胺类抗生素、庆大霉素、阿米卡星的耐药率高,对氟诺唑酮类抗生素及加酶头孢菌素较敏感。结论产酶是黄杆菌多重耐药的主要原因之一,头孢哌酮/他唑巴坦、头孢吡肟对其具有良好的抗菌活性。     

一株广泛耐药恶臭假单胞菌耐药机制研究

目的研究一株临床分离广泛耐药恶臭假单胞菌(extensively drug resistant Pseudomonas putida,XDR-PP)的耐药机制,以期指导临床合理使用抗菌药物。方法采用VITEK-2compact全自动微生物分析系统对菌株进行鉴定及药敏试验;改良Hodge试验初筛碳青霉烯酶;EDTA-亚胺培南协同试验和亚胺培南双纸片增效法初筛金属酶;PCR检测16SrRNA甲基化酶基因、金属β-内酰胺酶基因及可移动性遗传元件基因,产物测序,结果经BLAST软件比对分析。结果该菌株对所检测的抗菌药物均耐药。改良Hodge试验、EDTA-亚胺培南协同试验和亚胺培南双纸片增效法结果均为阳性。基因检测结果显示,金属β-内酰胺酶基因阳性的有blaVIM-2和blaIMP-4两种基因,16SrRNA甲基化酶基因阳性的有armA基因,13种可移动性遗传元件中,intI、tnpU和merA基因阳性,其余均为阴性。结论blaVIM-2、blaIMP-4、armA、intI、tnpU以及merA基因是导致其广泛耐药的重要机制。恶臭假单胞菌中同时检出blaVIM-2和blaIMP-4两种金属β-内酰胺酶基因及armA基因为国内首次报道。     

ICU多重耐药菌的分布与耐药性分析

目的探讨ICU病区多重耐药菌(Multidrug resistant organism,MDRO)的分布和耐药性,为ICU患者抗感染治疗和医院感管工作提供科学依据。方法使用法国梅里埃ATB-Expression鉴定药敏分析仪完成微生物鉴定和药敏工作,回顾性分析ICU患者2011年1月至2013年12月的培养鉴定结果和药敏结果,利用WHONET 5.4软件对数据进行分析。结果共分离MDRO 432株,检出率居前三位的分别是:耐碳青霉烯类抗菌药物鲍曼不动杆菌(CR-AB,54.2%)、产超广谱β-内酰胺酶(ESBLs)细菌(23.8%)、多重耐药/泛耐药铜绿假单胞菌(MDR/PDR-PA,12.5%)。耐碳青霉烯类抗菌药物肠杆菌科细菌(CRE)的分离率逐年上升,未检出耐万古霉素肠球菌(VRE)。结论 MDRO是我院ICU病区医院感染的主要病原菌,临床应加强目标监测,控制和减少多重耐药菌在院内流行。