多重耐药鲍曼不动杆菌消毒剂耐药基因检测及同源性分析

目的探讨多重耐药鲍曼不动杆菌（MDRAB）临床分离株对医院常用消毒剂苯扎溴铵和醋酸氯已定的耐药表型与qacE△1-sul1基因型的相关性并分析菌株的同源性。方法用微量肉汤稀释法检测菌株的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）,用PCR方法检测qacE△1-sul1基因,用脉冲场凝胶电泳（PFGE）分析菌株的同源性。结果20株MDRAB中,18株（90％）qacE△l-sul1阳性,2株（10％）qacE△1-sul1阴性。qacE△1-sul1阳性株对苯扎溴铵的MIC50、MIC90、MBC50和MBC90分别是qacE△1-sul1阴性株的2倍、2倍、8倍和8倍。而qacE△1-sul1阳性株对醋酸氯已定的MIC50、MIC90、MBC50和MBC90分别是qacE△1-sul1阴性株的2倍、2倍、4倍和8倍。PF-GE显示：18株qacE△1-sul1阳性MDRAB可分为A～F6个PFGE克隆。2株qacE△1-sul1阴性MDRAB均为B克隆。结论MDRAB对消毒剂耐药性升高与qacE△1-sul1基因相关,临床存在多个克隆株的传播。     

Dystroglycan and protein O-mannosyltransferases 1 and 2 are required to maintain integrity of Drosophila larval muscles

In vertebrates, mutations in Protein O-mannosyltransferase1 (POMT1) or POMT2 are associated with muscular dystrophy due to a requirement for O-linked mannose glycans on the Dystroglycan (Dg) protein. In this study we examine larval body wall muscles of Drosophila mutant for Dg, or RNA interference knockdown for Dg and find defects in muscle attachment, altered muscle contraction, and a change in muscle membrane resistance. To determine if POMTs are required for Dg function in Drosophila, we examine larvae mutant for genes encoding POMT1 or POMT2. Larvae mutant for either POMT, or doubly mutant for both, show muscle attachment and muscle contraction phenotypes identical to those associated with reduced Dg function, consistent with a requirement for O-linked mannose on Drosophila Dg. Together these data establish a central role for Dg in maintaining integrity in Drosophila larval muscles and demonstrate the importance of glycosylation to Dg function in Drosophila. This study opens the possibility of using Drosophila to investigate muscular dystrophy.     

呼吸内科重症监护室一起多重耐药鲍曼不动杆菌感染暴发的调查

目的及时发现医院感染暴发苗头,查找感染源,切断传播途径,最大限度减少医院感染发生。方法对2009年4月26～28日期间RICU发生的4例鲍曼不动杆菌感染病例进行聚集感染危险因素调查,采集标本进行目标菌检测,应用ERIC—PCR技术对聚集病例和散发病例菌株进行基因同源性分析,采取综合性防控措施进行医院感染具体干预。结果4例患者确定为医院感染病例,4例患者均有气管插管操作经历,3例患者环境卫生学检测存在耐药谱相同的鲍曼不动杆菌,ERIC—PCR分析4例患者检出的鲍曼不动杆菌具有基因同源性,确定为医院感染暴发。严格执行隔离防护措施和后续加强上呼吸机患者管理,未再出现聚集病例。结论医院感染暴发危及患者医疗安全,应用分子生物学分析手段可以促进医院感染防控措施的效果;加强上呼吸机患者的管理,加强手卫生的管理可以有效防控医院感染聚集暴发。     

鲍曼不动杆菌的耐药性检测及泛耐药菌株的耐药基因研究

目的考察临床分离的鲍曼不动杆菌的耐药性并对泛耐药菌株的耐药基因进行检测。方法用纸片扩散法对60株临床分离鲍曼不动杆菌进行药物敏感试验,并用PCR法检测8株泛耐药菌株携带7种耐药基因OXA-51、OXA-23、OXA-24、OXA-58、aac(3)-Ⅰ、gyr A、abe M的情况。结果所分离的鲍曼不动杆菌对临床常用的β-内酰胺类抗生素、氨基糖苷类抗生素、四环素类抗生素、磺胺类抗菌药及喹诺酮类抗菌药均产生耐药性;在8株泛耐药鲍曼不动杆菌中OXA-51、OXA-23、OXA-58、gyr A、abe M基因检测均全部呈阳性;OXA-24基因检测均呈阴性;aac(3)-I基因检测有5株呈阳性,3株呈阴性。结论鲍曼不动杆菌耐药情况严重,其耐药机制与多种耐药基因密切相关。     

Proteomic analysis of a fraction enriched in cell envelope proteins of Acinetobacter baumannii

Acinetobacter baumannii is a multiresistant opportunistic nosocomial pathogen. A protein fraction was purified and analyzed by 2-DE. Twenty-nine major protein spots were selected for protein identification using trypsin digestion and MS analysis. As the A. baumannii genome has not yet been described, protein identification was performed by homology with other Acinetobacter species in the NCBi database. We identified ribosomal proteins, chaperones, elongation factors and outer membrane proteins (Omp), such as OmpA and the 33-36-kDa OMP. Proteomic analysis of A. baumannii provides a platform for further studies in antimicrobial resistance.     

Multidrug-resistant genes are associated with an 86-kb island in Acinetobacter baumannii

A strain of multidrug-resistant Acinetobacter baumannii that caused a 26% mortality rate in a French epidemic was characterized and compared with an antibiotic-susceptible strain of A. baumannii. The multiresistant strain carries many of its antibiotic-resistance genes on an 86-kb region, whereas the susceptible strain lacks these genes in a homologous region. Characterization of this multidrug-resistant A. baumannii strain highlights the limited options for current therapy and raises concerns for future treatment options in an era in which few novel antibiotics are being developed.     

Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.     

Extracellular leucine-rich repeat proteins are required to organize the apical extracellular matrix and maintain epithelial junction integrity in C. elegans

Epithelial cells are linked by apicolateral junctions that are essential for tissue integrity. Epithelial cells also secrete a specialized apical extracellular matrix (ECM) that serves as a protective barrier. Some components of the apical ECM, such as mucins, can influence epithelial junction remodeling and disassembly during epithelial-to-mesenchymal transition (EMT). However, the molecular composition and biological roles of the apical ECM are not well understood. We identified a set of extracellular leucine-rich repeat only (eLRRon) proteins in C. elegans (LET-4 and EGG-6) that are expressed on the apical surfaces of epidermal cells and some tubular epithelia, including the excretory duct and pore. A previously characterized paralog, SYM-1, is also expressed in epidermal cells and secreted into the apical ECM. Related mammalian eLRRon proteins, such as decorin or LRRTM1-3, influence stromal ECM or synaptic junction organization, respectively. Mutants lacking one or more of the C. elegans epithelial eLRRon proteins show multiple defects in apical ECM organization, consistent with these proteins contributing to the embryonic sheath and cuticular ECM. Furthermore, epithelial junctions initially form in the correct locations, but then rupture at the time of cuticle secretion and remodeling of cell-matrix interactions. This work identifies epithelial eLRRon proteins as important components and organizers of the pre-cuticular and cuticular apical ECM, and adds to the small but growing body of evidence linking the apical ECM to epithelial junction stability. We propose that eLRRon-dependent apical ECM organization contributes to cell-cell adhesion and may modulate epithelial junction dynamics in both normal and disease situations.     

POT1 and TRF2 cooperate to maintain telomeric integrity

Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2(DeltaBDeltaM), bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2(DeltaBDeltaM)-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.     

The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii

ISAba1 was found in all widespread clones of Acinetobacter baumannii in the United Kingdom. All isolates studied had a blaOXA-51-like carbapenemase gene; some also had blaOXA-23-like and/or blaOXA-58-like. Among isolates with blaOXA-51-like as sole carbapenemase gene, only those with ISAba1 adjacent to blaOXA-51-like were carbapenem resistant. Minor differences in blaOXA-51-like sequence were observed in resistant and susceptible isolates. Isolates with blaOXA-23-like in addition were consistently resistant to carbapenems; in all of these ISAba1 lay upstream of blaOXA-23-like, but was not associated with blaOXA-51-like. These results suggest that ISAba1 is providing the promoter for blaOXA-51-like and, probably, for blaOXA-23-like.     

Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts

In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 alpha (LAP2alpha) upon entry and exit from G(0) is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2alpha are down-regulated in G(0). Although RbS780 phosphoform and LAP2alpha are up-regulated upon reentry into G(1) and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2alpha is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G(1) phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2alpha or lamin A/C in HDFs leads to accumulation of Rb in speckles and G(1) arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2alpha and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.     

Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability

Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.     

The SPIRAL genes are required for directional control of cell elongation in Aarabidopsis thaliana

Cells at the elongation zone expand longitudinally to form the straight central axis of plant stems, hypocotyls and roots, and transverse cortical microtubule arrays are generally recognized to be important for the anisotropic growth. Recessive mutations in either of two Arabidopsis thaliana SPIRAL loci, SPR1 or SPR2, reduce anisotropic growth of endodermal and cortical cells in roots and etiolated hypocotyls, and induce right-handed helical growth in epidermal cell files of these organs. spr2 mutants additionally show right-handed twisting in petioles and petals. The spr1spr2 double mutant's phenotype is synergistic, suggesting that SPR1 and SPR2 act on a similar process but in separate pathways in controlling cell elongation. Interestingly, addition of a low dose of either of the microtubule-interacting drugs propyzamide or taxol in the agar medium was found to reduce anisotropic expansion of endodermal and cortical cells at the root elongation zone of wild-type seedlings, resulting in left-handed helical growth. In both spiral mutants, exogenous application of these drugs reverted the direction of the epidermal helix, in a dose-dependent manner, from right-handed to left-handed; propyzamide at 1 microM and taxol at 0.2-0.3 microM effectively suppressed the cell elongation defects of spiral seedlings. The spr1 phenotype is more pronounced at low temperatures and is nearly suppressed at high temperatures. Cortical microtubules in elongating epidermal cells of spr1 roots were arranged in left-handed helical arrays, whereas the highly isotropic cortical cells of etiolated spr1 hypocotyls showed microtubule arrays with irregular orientations. We propose that a microtubule-dependent process and SPR1/SPR2 act antagonistically to control directional cell elongation by preventing elongating cells from potential twisting. Our model may have implicit bearing on the circumnutation mechanism.     

Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.