miR-26抑制帕金森患者泛素-蛋白酶体系统对含α突触核蛋白降解作用研究

目的 探讨miR-26抑制帕金森患者泛素-蛋白酶体系统(UPS)对含α突触核蛋白(α-Syn)降解作用.方法 将120只成年雄性C57BL/6小鼠随机分为对照组与模型组,每组各60只.通过1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)法建立帕金森病小鼠模型,采用反转录酶-聚合酶链锁反应(RT-PCR)检测模型...     

日本血吸虫重组两歧双歧杆菌(pGEX-Sj26GST)疫苗的构建及鉴定

目的 构建日本血吸虫重组两歧双歧杆菌（pGEX-Sj26GST）疫苗,并对其进行鉴定.方法 采用RNeasy Mini试剂盒提取日本血吸虫成虫总RNA,采用逆转录聚合酶链反应（RT-PCR）方法获得Sj26GST抗原编码基因;将该编码基因与pGEX-1λT载体进行连接,得到重组质粒pGEX-Sj26GST,并对其进行双酶切鉴定;将重组质粒电转化入两歧双歧杆菌中,构建重组两歧双歧杆菌（pGEX-Sj26GST）疫苗,PCR鉴定疫苗.结果 RT-PCR扩增出的日本血吸虫成虫Sj26GST基因片段长度为676 bp;双酶切证实Sj26GST抗原编码基因成功插入pGEX-1λT载体中;PCR证实从重组两歧双歧杆菌疫苗中扩增出长度为676 bp 的Sj26GST基因片段.结论 成功构建了日本血吸虫重组两歧双歧杆菌（pGEX-Sj26GST）疫苗.     

13-三体综合征胎儿的孕中期超声指标分析

目的 评估孕中期超声检查对13-三体综合征胎儿的诊断价值。方法 回顾分析本院26胎经染色体核型分析确诊为13-三体综合征胎儿的声像图资料。结果 前脑无裂畸形12胎(12/26,46.15%)、唇/腭裂9胎(9/26,34.62%)、室间隔缺损9胎(9/26,34.62%)、肾脏异常8胎(8/26,30.77%)、眼眶异常7胎(7/26,26.93%)、鼻发育异常5胎(5/26,19.23%)、脑室增宽4胎(4/26,15.38%)、羊水过多伴宫内生长受限2胎(2/26,7.69%)、独眼1胎(1/26,3.85%)。声像图异常率为92.31%(24/26),均表现为2种及以上异常指标。结论 孕中期超声检查对诊断13-三体综合征具有一定的意义;结合染色体核型分析可提高13-三体综合征的产前诊断率,并能降低其出生率。     

Long noncoding RNA CCDC26 as a potential predictor biomarker contributes to tumorigenesis in pancreatic cancer

Pancreatic cancer (PC) is the fourth most common cancer worldwide and has the least patient survival rate of any cancer. Emerging studies have demonstrated that long noncoding RNAs (lncRNAs) were present in cancer patients and have shown great potential as powerful markers and therapeutic targets. However, little is known about the role of lncRNAs in PC. The present study aimed to investigate the expression pattern, clinical significance and biological function of lncRNA CCDC26 (CCDC26) in PC. With quantitative real-time PCR, we analyzed CCDC26 expression levels in 40 PC patients. We found that the CCDC26 expression was significantly higher in PC tissues than in normal tissues. CCDC26 levels were correlated with tumor size, tumor number, and reduced overall survival (OS). Univariate and multivariate analysis showed that CCDC26 expression is an independent prognostic factor of OS in patients with PC. Additionally, ROC^AUC of CCDC26 was up to 0.663, implicating that CCDC26 could be a diagnostic marker for distinguishing PC from normal. Knockdown of CCDC26 expression by small interfering RNA significantly promoted growth arrest and apoptosis. Moreover, we found that the expression of CCDC26 was positively correlated with PCNA and Bcl2. Our data suggest that CCDC26 may be identified as a novel oncogene in PC, and responsible for growth and apoptosis of cancer cell, partly by regulating the PCNA and Bcl2 expression. This work provides a novel biomarker and therapeutic target of PC for cancer clinic in future.     

超声心动图诊断右肺动脉异常起源

目的 探讨经胸超声心动图（TTE）诊断单侧右肺动脉异常起源（AORPA）的价值。方法 收集经手术确诊为AORPA的患儿26例,分析其TTE特征。结果 AORPA的TTE表现为右肺动脉异常起源于升主动脉,主肺动脉和左肺动脉正常显示。26例患儿TTE均明确诊断为AORPA,诊断符合率为100%。其中9例合并Berry综合征,1例合并主动脉缩窄,22例合并动脉导管未闭,23例合并继发孔型房间隔缺损或卵圆孔未闭,25例合并重度肺动脉高压,TTE对各合并结构异常的诊断准确率分别为100%（26/26）、100%（26/26）、96.15%（25/26）、92.31%（24/26）、100%（26/26）。结论 TTE可早期、准确诊断AORPA,对其他心内伴随畸形诊断准确率高,多切面扫查有助于减少漏、误诊,可作为AORPA的首选检查方法。     

miR-26a靶向调控AP-2γ表达对神经母细胞瘤细胞增殖的影响

[目的]探讨miR-26a是否可通过靶向调控AP-2γ表达而抑制神经母细胞瘤细胞增殖.[方法]通过双荧光素酶基因分析检测miR-26a对AP-2γ 3＇非编码区（3＇UTR）-荧光素酶的影响;采用Western blot方法检测miR-26a模拟物转染神经母细胞瘤细胞中AP 2γ表达水平;将AP-2γ shRNA转染SK-N AS细胞,MTS细胞增殖活性检测分析干扰AP-2γ表达对神经母细胞瘤细胞增殖能力.[结果]双荧光素酶活性检测显示miR-26a特异性地与AP-2γ的3＇UTR结合,抑制其荧光素酶活性.过表达miR-26a的神经母细胞瘤细胞AP2γ蛋白表达水平降低;shRNA干扰AP-2γ表达能抑制SK-N-AS细胞的增殖能力.[结论]miR-26a通过靶向调控AP-2γ表达而抑制神经母细胞瘤细胞的增殖.     

Activation of lymphocytes by anti-CD3 single-chain antibody dimers expressed on the plasma membrane of tumor cells

Activation of cytotoxic T cells without MHC restriction was attempted by expressing single-chain antibodies (scFv) against CD3 on the surface of tumor cells. A chimeric protein consisting of a scFv of mAb 145.2C11, the hinge-CH2-CH3 region of human IgG1, and the transmembrane and cytosolic domains of murine CD80 formed disulfide-linked dimers on the plasma membrane of cells and specifically bound lymphocytes. Anti-CD3 scFv dimers expressed on the cell surface induced CD25 (IL-2 receptor alpha-chain) expression and proliferation of splenocytes. CT26 tumor cells engineered to express surface scFv dimers (CT26/2C11) also induced potent lymphocyte cytotoxicity with or without addition of exogenous IL-2. Splenocytes activated by CT26/2C11 cells also killed wild-type CT26 cells, indicating that activated splenocytes could kill bystander tumor cells. Immunization of BALB/c mice with irradiated CT26/2C11 cells did not protect against a lethal challenge of CT26 cells, suggesting that systemic immunity was not induced. However, the growth of CT26 tumors containing 50% CT26/2C11 cells was significantly retarded compared with CT26 tumors, whereas CT26/2C11 tumors did not grow in syngeneic mice. These results suggest that expression of anti-CD3 scFv dimers on tumors may form the basis for a novel therapeutic strategy for tumors that exhibit defects in antigen processing or presentation. Gene Therapy (2000) 7, 339-347.     

Slc26a6敲除对小鼠胰腺导管上皮细胞Slc26a3和CFTR表达水平的影响

目的通过检测Slc26a6敲除型和野生型小鼠离体胰腺导管CFTR和Slc26a3表达的差异,进一步探讨胰腺导管上皮细胞腔面膜上CFTR和Slc26s的相关性。方法从野生型和Slc26a6敲除小鼠提取胰腺组织并剥离出离体胰腺导管,提取RNA,逆转录生成cDNA,进行相对定量Real-Time PCR实验,以-βactin作为内参基因,野生型小鼠肾脏目的基因含量作为对照组,目的基因的相对定量按公式得出：目的基因=2-（△△Ct）。结果野生型小鼠Slc26a3的相对表达量为7.84±0.22,Slc26a6敲除小鼠Slc26a3的相对表达量为7.37±0.36,二者差异无显著性（P〉0.05）。野生型小鼠CFTR的相对表达量为0.08±0.003,Slc26a6敲除小鼠CFTR的相对表达量为0.05±0.003,二者差异无显著性（P〉0.05）。结论 Slc26a6敲除对胰腺导管上皮细胞Slc26a3和CFTR的表达水平无显著影响,故腔面膜上Slc26a6对CFTR的增强作用可能并不是通过增加其表达水平实现的;另外,Slc26a3可能参与了小鼠胰腺导管上皮细胞HCO3-的分泌,但与Slc26a6在功能上的相关性有待进一步研究。     

CD26/dipeptidyl peptidase IV: A comparative study of healthy persons and kidney transplant recipients before and early after transplantation

Objectives

Human CD26 is co-stimulatory for lymphocytes, circulates in a soluble form in blood (sCD26), and has intrinsic dipeptidyl peptidase IV (DPPIV) activity. Associations between CD26 expression on the surface of T cells (CD26 +/CD3 +) and acute rejection and between (CD26 +/CD3 +)/DPPIV and clinical immunosuppression have been reported. These results encouraged the investigation of CD26 as a potential biomarker to optimize immunosuppressive therapy. To better understand the significance of CD26, a comparative study of CD26 expression on CD3 + cells, sCD26 concentration, and DPPIV activity in healthy persons (HP) and kidney transplant recipients (KTR) was performed.

Design and methods

Thirty-one HP and 34 KTR were included in the study. CD26 +/CD3 + was determined by FACS, sCD26 concentration was determined by ELISA, and DPP activity was determined by spectrophotometry. For KTR, these parameters were studied on the day before transplantation (preTx) and 7 ± 1 days after transplantation (postTx).

Results

There was no significant difference in the CD26 +/CD3 +, sCD26, and DPPIV data regarding gender, donor type (16 living donors), delayed graft function (n = 8), or presence of ≥ 4HLA mismatches (n = 16). Compared to the HP data, preTx CD26 +/CD3 + was 4.5-fold higher, sCD26 was 1.2-fold higher, and DPPIV showed no significant difference. PostTx, CD26 +/CD3 + was 3.8-fold higher, and sCD26 and DPPIV decreased significantly, reaching lower values than that observed in HP. Re-transplanted patients (n = 5) showed significantly lower preTx CD26 +/CD3 + expression than patients receiving their first transplant. Patients with preemptive transplantation (n = 7) showed higher postTx CD26 +/CD3 + expression than patients on dialysis.

Conclusions

CD26 expression on CD3 + cells was strongly increased in patients with end stage kidney disease compared to HP and remained high early postTx. The differences in sCD26 and DPPIV behavior compared to that of CD26 +/CD3 + postTx may reflect a regulatory response to the new immunological situation and the effects of therapy.     

超声心动图评价偏心性主动脉瓣反流所致室壁冲击伤

目的 观察经胸彩色多普勒超声心动图评价偏心性主动脉瓣反流（AR）所致室壁冲击伤的效果。方法 26例经胸彩色多普勒超声显示中量及以上偏心性AR患者反流束已明确冲击至室间隔或左心室游离壁某处并发生变形，分析其影像学资料，观察偏心性AR对室壁造成冲击伤的形态学特点。结果 偏心性AR高速射流冲击部位均位于室间隔或左心室游离壁基底段或中上段（26/26，100%）。其中4例（4/26，15.38%）下间隔和下壁均被冲击，5例（5/26，19.23%）仅下壁被冲击，13例（13/26，50.00%）下壁和下侧壁均被冲击，2例（2/26，7.69%）仅下侧壁被冲击，下间隔、下壁和下侧壁均被冲击者2例（2/26，7.69%）。被冲击室壁处均可见局部扩张（26/26，100%）；6例（6/26，23.08%）运动幅度明显减低或消失，10例（10/26，38.46%）轻度减低，10例（10/26，38.46%）正常。CMR图像上除可见局部室壁扩张和运动减低外，严重者延迟增强时可见相应受损部位心肌纤维化改变。核素心肌灌注检查中，冲击伤重者可见局部心肌血流灌注和代谢减低均减低，而轻者无局部心肌血流灌注和代谢减低。结论 彩色多普勒超声心动图可用于观察中量及以上偏心性AR直接冲击室壁所致损伤部位和程度。     

Salt-Resistant Alpha-Helical Cationic Antimicrobial Peptides

Analogues based on the insect cecropin-bee melittin hybrid peptide (CEME) were studied and analyzed for activity and salt resistance. The new variants were designed to have an increase in amphipathic alpha-helical content (CP29 and CP26) and in overall positive charge (CP26). The alpha-helicity of these peptides was demonstrated by circular dichroism spectroscopy in the presence of liposomes. CP29 was shown to have activity against gram-negative bacteria that was similar to or better than those of the parent peptides, and CP26 had similar activity. CP29 had cytoplasmic membrane permeabilization activity, as assessed by the unmasking of cytoplasmic beta-galactosidase, similar to that of CEME and its more positively charged derivative named CEMA, whereas CP26 was substantially less effective. The activity of the peptides was not greatly attenuated by an uncoupler of membrane potential, carbonyl cyanide-m-chlorophenylhydrazone. The tryptophan residue in position 2 was shown to be necessary for interaction with cell membranes, as demonstrated by a complete lack of activity in the peptide CP208. Peptides CP29, CEME, and CEMA were resistant to antagonism by 0.1 to 0.3 M NaCl; however, CP26 was resistant to antagonism only by up to 160 mM NaCl. The peptides were generally more antagonized by 3 and 5 mM Mg2+ and by the polyanion alginate. It appeared that the positively charged C terminus in CP26 altered its ability to permeabilize the cytoplasmic membrane of Escherichia coli, although CP26 maintained its ability to kill gram-negative bacteria. These peptides are potential candidates for future therapeutic drugs.     

Comparison of nine resistance interpretation systems for HIV-1 genotyping

HIV-1 genotyping has become a widely accepted tool for monitoring antiretroviral therapy. However, the benefit of genotyping is limited by the need to interpret the mutation pattern in order to obtain a prediction regarding susceptibility to each antiretroviral drug. Several different interpretation systems are currently available, commercially or free of charge; some are in combination with the genotyping test system used. In this study, we compared the results obtained on patient samples, using nine different resistance interpretation systems for HIV-1 genotype, and identified mutation patterns responsible for discordances between these systems. HIV-1 genotypes from 26 patients were obtained using the ViroSeq HIV-1 Genotyping System (Applied Biosystems). Nine resistance interpretation systems were used: the 'virtual phenotype' systems VirtualPhenotype (Virco) and Geno2Pheno (http://cartan.gmd.de/geno2pheno.html), the rule-based resistance algorithms Antiretroviral Drug Resistance Report (Applied Biosystems), Stanford HIV-SEQ program (http://hivdb.stanford.edu/) and the ViroScorer system (ABL; including ANRS AC11, Detroit Medical Center, Grupo de Aconselhamento Virológico, CHL, and Rega). Discordance was defined as the same sequence being interpreted as resistant and sensitive to a substance by different systems, with intermediate scores being regarded as neutral. Results for lopinavir were not available from some interpretation systems. None of the 26 patient samples had concordant results for all drugs. The highest degree of concordance was found for the resistance scoring of lamivudine (25/26), followed by nelfinavir (23/26), indinavir, ritonavir, saquinavir (all 22/26), zidovudine, nevirapine (all 21/26), lopinavir, efavirenz (all 18/26), amprenavir, delavirdine (all 17/26), stavudine, abacavir (all 13/26), zalcitabine (7/26) and didanosine (5/26). In most cases, it was only one interpretation system that gave an interpretation different from the others. If this interpretation system was omitted, the overall discordance rate decreased by a statistically significant degree. There exists, therefore, an urgent need for standardization of interpretation algorithms for HIV-1 genotyping.     

Design and evaluation of S-nitrosylated human serum albumin as a novel anticancer drug

In recent studies, the cytotoxic activity of NO has been investigated for its potential use in anticancer therapies. Nitrosated human serum albumin (NO-HSA) may act as a reservoir of NO in vivo. However, there are no published reports regarding the effects of NO-HSA on cancer. Therefore, the present study investigated the antitumor activity of NO-HSA. NO-HSA was prepared by incubating HSA, which had been sulfhydrylated using iminothiolane, with isopentyl nitrite (6.64 mol NO/mol HSA). Antitumor activity was examined in vitro using murine colon 26 carcinoma (C26) cells and in vivo using C26 tumor-bearing mice. Exposure to NO-HSA increased the production of reactive oxygen species in C26 cells. Flow cytometric analysis using rhodamine 123 showed that NO-HSA caused mitochondrial depolarization. Activation of caspase-3 and DNA fragmentation were observed in C26 cells after incubation with 100 muM NO-HSA for 24 h, and NO-HSA inhibited the growth of C26 cells in a concentration-dependent manner. The growth of C26 tumors in mice was significantly inhibited by administration of NO-HSA compared with saline and HSA treatment. Immunohistochemical analysis of tumor tissues demonstrated an increase in terminal deoxynucleotidyl transferase dUTP nickend labeling-positive cells in NO-HSA-treated mice, suggesting that inhibition of tumor growth by NO-HSA was mediated through induction of apoptosis. Biochemical parameters (such as serum creatinine, blood urea nitrogen, aspartate aminotransferase, and alanine aminotransferase) showed no significant differences among the three treatment groups, indicating that NO-HSA did not cause hepatic or renal damage. These results suggest that NO-HSA has the potential for chemopreventive and/or chemotherapeutic activity with few side effects.     

Strongylophorine-26, a Rho-dependent inhibitor of tumor cell invasion that reduces actin stress fibers and induces nonpolarized lamellipodial extensions

Strongylophorine-26, a new meroditerpenoid, was recently identified as an inhibitor of cancer cell invasion. This study was undertaken to characterize its mechanism of action. We find that strongylophorine-26 inhibits the motility of MDA-MB-231 breast carcinoma cells on a plastic surface. Upon addition of strongylophorine-26, rapid cell contraction and depolarization occurred, followed by spreading and flattening of the entire cell. Treated cells exhibited increased membrane ruffling throughout and extended lamellipodia in all directions. Strongylophorine-26 induced a decrease in actin stress fibers, a dramatic increase in the size and number of focal adhesions, and the appearance of a dense meshwork of actin filaments around the cell periphery. Strongylophorine-26 caused a transient activation of the small GTPase Rho and treatment with the Rho inhibitor C3 exoenzyme abrogated the anti-invasive activity of strongylophorine-26. These effects are distinct from those of many motility and angiogenesis inhibitors that seem to act by a common mechanism involving the induction of actin stress fibers. This difference in mechanism of action sets strongylophorine-26 apart as an experimental anticancer agent and indicates that pharmacologic inhibition of cell migration may be achieved by mechanisms not involving the stabilization of actin stress fibers.     

Biotin delivery to brain with a covalent conjugate of avidin and a monoclonal antibody to the transferrin receptor.

The OX26 mouse monoclonal antibody to the rat transferrin receptor undergoes transcytosis through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, owing to high concentrations of transferrin receptor on the BBB. This property allows the OX26 antibody to serve as a brain drug transport vector. To simplify coupling of therapeutics to the OX26 antibody, the present studies examine the use of the avidin/biotin system to promote coupling of biotin and biotinylated drugs to brain transport vectors. The OX26 antibody was affinity purified from ascites fluid and was covalently coupled via a thioether linkage to avidin, and the conjugate was purified to homogeneity by gel filtration fast protein liquid chromatography. The biotin binding capacity of the avidin-OX26 conjugate was measured, and 2.3 biotin binding sites per avidin-OX26 (1:1) conjugate were detected. Transcytosis through the BBB of [3H]biotin bound to either unconjugated avidin or to the avidin-OX26 conjugate was measured with an internal carotid artery perfusion/capillary depletion technique. [3H]Biotin bound to the avidin-OX26 conjugate was transported through the BBB at rates that equaled the rate of transcytosis of the unconjugated OX26 antibody. The clearance from plasma of [3H]biotin bound to the avidin-OX26 conjugate approximated the rate of clearance of the unconjugated OX26 antibody, and not the rate of clearance of [3H]biotin bound to avidin, which was cleared from plasma at much faster rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

How accurately can the aetiology of cardiac arrest be established in an out-of-hospital setting? Analysis by "concordance in diagnosis crosscheck tables"

Introduction

Several previous studies have focused on establishing the cause of cardiac arrest (CA) during cardiopulmonary resuscitation (CPR) provided in an out-of-hospital setting.

Objectives

To analyze the ability of professional advanced life support providers to correctly establish the aetiology of cardiac arrest during out-of-hospital CPR.

Study design

A retrospective cohort study analysing 211 cases of out-of-hospital cardiac arrest.

Method

The aetiology assumed by out-of-hospital physicians was compared with the diagnosis that was later established by clinicians or pathologists.

Results

Cases were sorted into five diagnostic groups and the overall diagnostic concordance was 74.4% (157 of 211 cases). The cardiac aetiology was presumed in 132 out of 211 patients and confirmed in 135 out of 211 patients.However, an analysis of individual cases of the cardiac causes of cardiac arrest revealed diagnostic matches in only 112 cases. Acute myocardial infarction (AMI) or pulmonary embolism (PE), both of which represent cases that can be potentially influenced by thrombolytic therapy, were presumed in 74 (53 + 21) and confirmed in 97 (77 + 20) cases, however with individual diagnostic matches in only 55 cases.

Conclusion

This study demonstrates the importance of analysing concordance in presumed and definitive diagnosis of individual cases, since an overall comparison in a cohort of cases may be highly misleading. It introduces the method of the crosscheck table for visualization and comparison of presumed and final diagnoses. The two alternative approaches of inclusion rule for applying the thrombolytic therapy in out-of-hospital care were discussed with regard to the recent TROICA study.     

Ac2-26 mitigated acute respiratory distress syndrome rats via formyl peptide receptor pathway

BackgroundAcute respiratory distress syndrome (ARDS) is characterized by severe local and systemic inflammation. Ac2-26, an Annexin A1 Peptide, can reduce the lung injury induced by reperfusion via the inhibition of inflammation. The present study aims to evaluate the effect and mechanism of Ac2-26 in ARDS.MethodsThirty-two rats were anaesthetized and randomized into four groups: sham (S), ARDS (A), ARDS/Ac2-26 (AA), and ARDS/Ac2-26/BOC-2 (AAB) groups. Rats in the S group received saline for intratracheal instillation, while rats in the other three groups received endotoxin for intratracheal instillation, in order to prepare the ARDS and inject the saline, Ac2-26, and Ac2-26 combined with BOC-2. After 24 h, the PaO₂/FiO₂ ratio was calculated. The lung tissue wet-to-dry weight ratio and the protein level in bronchoalveolar lavage fluid (BALF) were tested. Then, the cytokines in BALF and serum, and the inflammatory cells in BALF were investigated. Afterwards, the oxidative stress response and histological injury was evaluated. Subsequently, the epithelium was cultured and analyzed to estimate the effect of Ac2-26 on apoptosis.ResultsCompared to the S group, all indexes worsened in the A, AA, and AAB groups. Furthermore, compared to the S group, Ac2-26 significantly improved the lung injury and alveolar-capillary permeability, and inhibited the oxidative stress response. In addition, Ac2-26 reduced the local and systemic inflammation through the regulation of pro- and anti-inflammatory cytokines, and the decrease in inflammatory cells in BALF. Moreover, Ac2-26 inhibited the epithelium apoptosis induced by LPS through the modulation of apoptosis-regulated proteins. The protective effect of Ac2-26 on ARDS was partially reversed by the FPR inhibitor, BOC-2.ConclusionAc2-26 reduced the lung injury induced by LPS, promoted alveolar-capillary permeability, ameliorated the local and systemic inflammation, and inhibited the oxidative stress response and apoptosis. The protection of Ac2-26 on ARDS was mainly dependent on the FPR pathway.     

Characterization of OXA-25, OXA-26, and OXA-27, molecular class D beta-lactamases associated with carbapenem resistance in clinical isolates of Acinetobacter baumannii

Carbapenem resistance in Acinetobacter spp. is increasingly being associated with OXA-type beta-lactamases with weak hydrolytic activity against imipenem and meropenem. Such enzymes were characterized from Acinetobacter isolates collected in Belgium, Kuwait, Singapore, and Spain. The isolates from Spain and Belgium had novel class D beta-lactamases that were active against carbapenems. These were designated OXA-25 and OXA-26, respectively, and had >98% amino acid homology with each other and with the OXA-24 enzyme recently described by others from an Acinetobacter isolate collected elsewhere in Spain. The isolate from Singapore had OXA-27 beta-lactamase, another novel class D type with only 60% homology to OXA-24, -25, and -26, but with 99% homology to OXA-23 (ARI-1), described previously from an Acinetobacter baumannii isolate collected in Scotland. Sequence data were not obtained for the carbapenem-hydrolyzing OXA enzyme from the isolate from Kuwait; nevertheless, the enzyme was phenotypically similar to OXA-25 and -26. The enzymes OXA-23, -24, -25, -26, and -27 retained the STFK and SXV motifs typical of class D beta-lactamases, but the YGN motif was altered to FGN. The KTG motif was retained by OXA-27 and -23 but was replaced by KSG in OXA-24, -25, and -26. OXA-25 and -26 enzymes were strongly active against oxacillin, but unusually for an OXA-type beta-lactamase, OXA-27 had apparently weak activity, although measurement was complicated by biphasic kinetics. None of the new enzymes was transmissible to Escherichia coli recipients. Many Acinetobacter isolates are multiresistant to other antibiotics, and the emergence of class D enzymes with carbapenem-hydrolyzing activity is a disturbing development for antimicrobial chemotherapy.     

Studies on the mode of action of the antifungal hexapeptide PAF26

The small antimicrobial peptide PAF26 (Ac-RKKWFW-NH(2)) has been identified by a combinatorial approach and shows preferential activity toward filamentous fungi. In this work, we investigated the mode of action and inhibitory effects of PAF26 on the fungus Penicillium digitatum. The dye Sytox Green was used to demonstrate that PAF26 induced cell permeation. However, microscopic observations showed that sub-MIC concentrations of PAF26 produced both alterations of hyphal morphology (such as altered polar growth and branching) and chitin deposition in areas of no detectable permeation. Analysis of dose-response curves of inhibition and permeation suggested that growth inhibition is not solely a consequence of permeation. In order to shed light on the mode of PAF26 action, its antifungal properties were compared with those of melittin, a well-known pore-forming peptide that kills through cytolysis. While the 50% inhibitory concentrations and MICs of the two peptides against P. digitatum mycelium were comparable, they differed markedly in their fungicidal activities toward conidia and their hemolytic activities toward human red blood cells. Kinetic studies showed that melittin quickly induced Penicillium cell permeation, while PAF26-induced Sytox Green uptake was significantly slower and less efficient. Therefore, the ultimate growth inhibition and morphological alterations induced by PAF26 for P. digitatum are not likely a result of conventional pore formation. Fluorescently labeled PAF26 was used to demonstrate its specific in vivo interaction and translocation inside germ tubes and hyphal cells, at concentrations as low as 0.3 muM (20 times below the MIC), at which no inhibitory, morphological, or permeation effects were observed. Interestingly, internalized PAF26 could bind to cellular RNAs, since in vitro nonspecific RNA binding activity of PAF26 was demonstrated by electrophoretic mobility shift assays. We propose that PAF26 is a short, de novo-designed penetratin-type peptide that has multiple detrimental effects on target fungi, which ultimately result in permeation and killing.