Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1and AflMDR1 encode proteins of molecular weights 148 000 and 143 000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85 000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.     

结核分枝杆菌外排泵研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种传染病。随着多药耐药和广泛耐药结核分枝杆菌的出现,结核病的治疗变得更为艰难。近年来研究发现,结核分枝杆菌存在外排泵是其耐药的原因之一,现已发现结核分枝杆菌的主要易化子超家族(major facilitator superfamily,MFS)、三磷酸腺苷(adenosine-triphosphate,ATP)结合盒超家族(ATP-Binding Cassette,ABC)、耐受小节分裂区家族(resistance-nodulation-division,RND)和小耐多药性家族(small multidrug resistance,SMR)外排泵。但是人们对结核分枝杆菌外排泵介导的耐药现象认识不足,仍缺乏从新药发现角度研发外排泵抑制剂的研究。本文拟对结核分枝杆菌的ABC、MFS、RND和SMR外排泵的结构和功能,以及结核分枝杆菌外排泵抑制剂的研究进展进行综述。     

Relative contribution of target gene mutation and efflux to varying quinolone resistance in Irish Campylobacter isolates

The contribution of target gene mutations and active efflux to varying levels of quinolone resistance in Irish Campylobacter isolates was studied. The Thr-86-Ile modification of GyrA did not correlate with the level of quinolone resistance. The efflux pump inhibitor Phe-Arg-beta-Naphthylamide (PAbetaN) had no effect on the MICs to ciprofloxacin. In contrast, a PAbetaN sensitive efflux system contributed to the low-level nalixidic acid resistance phenotype. The lack of effect of PAbetaN in high-level resistant nalidixic isolates may be attributable to mutations identified in the CmeB efflux pump of these isolates. PAbetaN may have limited diagnostic value in the assessment of the contribution of efflux pump activity to ciprofloxacin resistance in Campylobacter.     

Propargylglycine-based antimicrobial compounds are targets of TolC-dependent efflux systems in Escherichia coli

A library of novel l-propargylglycine-based compounds were designed and synthesized with the goal of inhibiting the growth of Gram-negative bacteria by targeting LpxC, a highly conserved Gram-negative enzyme which performs an essential step in the lipid A biosynthetic pathway. These compounds were designed with and without a nucleoside and had varying tail structures, which modulate their lipophilicity. The synthetic scheme was improved compared to previous methods: a methyl ester intermediate was converted to a hydroxamic acid, which obviated the need for a THP protecting group and improved the yields and purity of the final compounds. Antimicrobial activity was observed for non-nucleoside compounds containing a phenyl propargyl ether tail (5) or a biphenyl tail (6). An MIC of 16 µg/mL was achieved for 6 in Escherichia coli, but inhibition was only possible in the absence of TolC-mediated efflux. Compound 5 had an initial MIC >160 µg/mL in E. coli. Enhancing outer membrane permeability or eliminating efflux reduced the MIC modestly to 100 µg/mL and 80 µg/mL, respectively. These results highlight the importance of hydrophobicity of this class of compounds in developing LpxC inhibitors, as well as the design challenge of avoiding multidrug efflux activity.     

Characterization of the antiseptic-resistance gene qacEΔ1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.     

Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance

A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition. Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30°C. The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics. Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity. Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially β-lactams. Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system. This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa. These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved. Received: December 18, 1997 / Accepted: March 16, 1998     

Low concentrations of medium-sized HDL particles predict incident CVD in chronic kidney disease patients

Patients with chronic kidney disease (CKD) are at high risk for CVD. However, traditional CVD risk factors cannot completely explain the increased risk. Altered HDL proteome is linked with incident CVD in CKD patients, but it is unclear whether other HDL metrics are associated with incident CVD in this population. In the current study, we analyzed samples from two independent prospective case-control cohorts of CKD patients, the Clinical Phenotyping and Resource Biobank Core (CPROBE) and the Chronic Renal Insufficiency Cohort (CRIC). We measured HDL particle sizes and concentrations (HDL-P) by calibrated ion mobility analysis and HDL cholesterol efflux capacity (CEC) by cAMP-stimulated J774 macrophages in 92 subjects from the CPROBE cohort (46 CVD and 46 controls) and in 91 subjects from the CRIC cohort (34 CVD and 57 controls). We tested associations of HDL metrics with incident CVD using logistic regression analysis. No significant associations were found for HDL-C or HDL-CEC in either cohort. Total HDL-P was only negatively associated with incident CVD in the CRIC cohort in unadjusted analysis. Among the six sized HDL subspecies, only medium-sized HDL-P was significantly and negatively associated with incident CVD in both cohorts after adjusting for clinical confounders and lipid risk factors with odds ratios (per 1-SD) of 0.45 (0.22–0.93, P = 0.032) and 0.42 (0.20–0.87, P = 0.019) for CPROBE and CRIC cohorts, respectively. Our observations indicate that medium-sized HDL-P—but not other-sized HDL-P or total HDL-P, HDL-C, or HDL-CEC—may be a prognostic cardiovascular risk marker in CKD.     

A Review of Phylogenetic and Metabolic Relationships Between the Acylamino Acids, N-Acetyl-l-Aspartic Acid and N-Acetyl-l-Histidine, in the Vertebrate Nervous System

Abstract: N -Acetyl- l -histidine (NAH) and N -acetyl- l -aspartic acid (NAA) are major constituents of vertebrate brain and eye with distinct phylogenetic distributions. They are characterized by high tissue concentrations, high tissue/extracellular fluid gradients, and a continuous regulated efflux into the extracellular fluid. As a result of parallel investigations over the past three decades, evidence has accumulated that suggests that the metabolism of NAA in the CNS of both homeothermic and poikilothermic vertebrates and the metabolism of NAH in the CNS of poikilothermic vertebrates are related. Tissue distribution and concentrations are similar, as well as timing of appearance during embryological development and their synthetic and degradative biochemistry. Both amino acids appear to be involved in a rapid tissue-to-fluid-space cycling phenomenon across a membrane. Evidence accumulating for each amino acid suggests a dynamic and important role in the CNS and the eye of vertebrates. A genetic disease in humans, Canavan's disease, is associated with NAA aciduria and aspartoacylase deficiency with concomitant accumulation of NAA and a spongy degeneration of the brain. In this article, evidence linking NAA and NAH metabolism is reviewed, and the hypothesis that NAA and NAH complement each other and are metabolic analogues involved with membrane transport is developed. Their enzyme systems also appear to exhibit plasticity in relation to osmoregulatory forces on an evolutionary time scale, with an apparent interface at the fish-tetrapod boundary.     

Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [μmol (g dry wt.)^–1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and α-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate. Received: 28 July 1997 / Accepted: 19 November 1997     

Effect of four heavy metals on the biology of Nostoc muscorum

Summary This study presents the effects of Cr, Pb, Ni and Ag on growth, pigments, protein, DNA, RNA, heterocyst frequency, uptake of NH₄ ⁺ and N0₃ ^–, loss of electrolytes (Na⁺ and K⁺), nitrate reductase and glutamine synthetase activities ofNostoc muscorum. The statistical tests revealed a direct positive correlation between the metal concentration and inhibition of different processes. Ni was found to be more toxic against growth, pigments and heterocyst differentiation compared to the other metals. Inhibition of pigment showed the following trend: chlorophyll > phycocyanin > carotenoid. No generalized trend for inhibition of macromolecules was observed. The loss of K⁺ and Na⁺ as affected by Cr, Ni and Pb was similar but more pronounced for K⁺ than Na⁺. The inhibition of physiological variables depicted the following trend: Na⁺ loss > K⁺ loss > glutamine synthetase > NH₄ uptake > growth > N0₃ ^– uptake > nitrate reductase > heterocyst frequency. This study therefore suggests that loss of electrolytes can be used as a first signal of metal toxicity in cyanobacteria. However, further study is needed to confirm whether the abnormality induced by nickel (branch formation) is a physiological or genetic phenomenon.