A C-terminal 18 amino acid deletion in MarR in a clinical isolate of Escherichia coli reduces MarR binding properties and increases the MIC of ciprofloxacin

As described recently, the different degree of fluoroquinolone resistance in a pair of sequential clinical isolates of Escherichia coli was due to the increased expression of the regulatory gene marA as a consequence of an 18 amino acid C-terminal deletion in the repressor MarR (MarRDelta). To further investigate the molecular mechanism of the loss of repressor function, we purified recombinant wild-type and mutated MarR, and tested their respective ability to form dimers and their specific DNA binding properties to the operator region marO. The dimerization capacity was analysed by non-reducing SDS-PAGE and by disuccinimidyl suberate-mediated cross-linking of the recombinant proteins. The binding of MarR was studied using the recombinant proteins and DNA probes containing the two identified binding sites in marO in the presence or absence of specific and non-specific DNA fragments. Dimerization of MarRDelta was reduced compared with MarR: the dimer portion was 33.8% (MarR) and 12.4% (MarRDelta) at a protein concentration of 10 muM. In mobility-shift assays MarRDelta showed a highly reduced complex formation. Footprinting analysis confirmed reduced binding of MarRDelta to its target sites, compared with MarR. The biochemical data are in full agreement with the crystal structure of MarR, which shows that the N- and C-terminal regions of MarR contribute to dimer formation. The data also indicate a major role of the MarR dimer as opposed to the monomer in DNA binding

Inhibition of wild-type human immunodeficiency virus and reverse transcriptase inhibitor-resistant variants by Phyllanthus amarus

Substantial progress has been made in research on natural products which effectively inhibit HIV-1 replication. Many active compounds were isolated from traditionally used medicinal plants including Phyllanthus species. This study shows that aqueous as well as alcohol-based Phyllanthus amarus extracts potently inhibit HIV-1 replication in HeLa CD4(+) cells with 50% effective concentration (EC50) values ranging from 0.9 to 7.6 mug/ml. A gallotannin enriched fraction showed enhanced activity (0.4 mug/ml), and the purified gallotannins geraniin and corilagin were most active (0.24 mug/ml). HIV-1 replication was also blocked in CD4(+) lymphoid cells with comparable EC50 values. Applying a cell-based internalization assay, we could demonstrate 70-75% inhibition of virus uptake at concentrations of 2.5 mug/ml for the water/alcohol extract and geraniin. In addition, a concentration-dependent inhibition of HIV-1 reverse transcriptase (RT) could be demonstrated in vitro. The 50% inhibitory concentration (IC50) values varied from 1.8 to 14.6 mug/ml. The ability to inhibit replication of a variety of RT inhibitor-resistant HIV-1 strains points to the potential of P. amarus extracts, as natural products, in the chemotherapy of HIV infections. (C) 2002 Elsevier Science B.V. All rights reserved

Concerted inhibitory activities of on HIV replication in vitro and ex vivo

Phyllanthus amarus derived preparations were previously shown to inhibit RT inhibitor-resistant HIV variants as efficiently as wildtype strains. The drugs target different steps of the HIV life cycle, thereby presenting multiple antiviral activities. Here we show that a water/alcohol extract blocks HIV-1 attachment and the HIV-1 enzymes integrase, reverse transcriptase and protease to different degrees. A gallotannin containing fraction and the isolated ellagitannins geraniin and corilagin were shown to be the most potent mediators of these antiviral activities. The P amarus derived preparations blocked the interaction of HIV-1 gp120 with its primary cellular receptor CD4 at 50% inhibitory concentrations of 2.65 (water/alcohol extract) to 0.48 mug/ml (geraniin). Inhibition was also evident for the HIV-1 enzymes integrase (0.48-0.16 mug/ml), reverse transcriptase (8.17-2.53 mug/ml) and protease (21.80-6.28 mug/ml). In order to prove the in vivo relevance of these biological activities, plant material was administered orally to volunteers and a potent anti-HIV activity in blood could be demonstrated. Sera at a final concentration of 5% reduced HIV replication by more than 30%. These results support the conclusion that P amarus has inhibitory effects on HIV not only in vitro but also in vivo. (C) 2004 Elsevier B.V. All rights reserved

Clinically significant borderline resistance of sequential clinical isolates of Klebsiella pneumoniae

Two sequential clinical isolates of Klebsiella pneumoniae (Kpn) were isolated from bronchoalveolar lavage fluid (Kpn#1) and sputum (Kpn#2) of a patient with pneumonia, complicated by anatomical and immunosuppressive problems due to Wegener's granulomatosis. Despite 4 weeks of systemic treatment with ciprofloxacin (CIP) Kpn#2 was isolated thereafter. A fluoroquinolone-resistant mutant (Kpn#1-SEL) was derived from Kpn#1 in vitro by selecting on agar plates supplemented with ofloxacin. Kpn#1, Kpn#1-SEL and Kpn#2 had an identical pattern in PFGE. CIP MICs were 0.25, 2 and 4 mg/l for Kpn#1, Kpn#2 and Kpn#1-SEL, respectively. Kpn ATCC 10031 (CIP MIC 0.002 mg/l) served as control. We analyzed mechanisms of fluoroquinolone resistance by deter-mining antibiotic susceptibility, organic solvent tolerance, accumulation of fluoroquinolones, dominance testing with wild-type topoisomerase genes (gyrA/B, parC/E), sequencing of the quinolone resistance determining regions of gyrA/B, parC/E and marR and Northern blotting of marR and acrAB genes. Compared with Kpn ATCC 10031, elevated MICs to fluoroquinolones and unrelated antibiotics in Kpn#1 was presumably due to a primary efflux pump other than AcrAB and increased the CIP MIC 125-fold. Although Kpn#1 tested sensitive according to NCCLS breakpoints, the elevated CIP MIC of 0.25 mg/l presumably rendered this isolate clinically resistant and lead to therapeutic failure in this case. Further increase of MIC to fluoroquinolones in vivo and in vitro was distinct. Kpn#1-SEL, selected in vitro, acquired a GyrA target mutation, whereas in Kpn#2 no known resistance mechanism could be detected. (C) 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved

Purification, cloning, and expression of a human enzyme with acyl coenzyme A: cholesterol acyltransferase activity, which is identical to liver carboxylesterase.

An enzyme with acyl coenzyme A:cholesterol acyltransferase (ACAT) activity was isolated from porcine liver, and sequences derived from trypsinized peptides indicated homology to liver carboxylesterase. By use of degenerate primers, human cDNA clones were identified, which were identical to human liver carboxylesterase. Expression of the full-length cDNA in Chinese hamster ovary (CHO) cells led to an approximately threefold increase in cellular ACAT activity. This was accompanied by an approximately 20-fold increase of cellular cholesteryl ester content. By light and electron microscopy, recombinant CHO cells contained numerous lipid droplets that were not present in control CHO cells. Expression of an antisense cDNA in HepG2 cells reduced cellular ACAT activity by 35% compared with control. To further investigate the role of the enzyme in cellular cholesterol homeostasis, regulation of the mRNA was investigated in 7-day cultured human mononuclear phagocytes (MNPs). When these cells were incubated in lipoprotein-deficient serum for 18 hours, the mRNA for ACAT/carboxylesterase was almost not detectable on Northern blots, whereas after incubation with acetylated low-density lipoproteins, a strong hybridization signal was obtained. This is evidence that the mRNA of ACAT/carboxylesterase is induced by cholesterol loading. It is concluded from the data presented that ACAT/carboxylesterase is relevant for cellular cholesterol esterification in vivo. The regulation in MNPs indicates that the enzyme is also involved in foam cell formation during early atherogenesis.</jats:p