Mutations in the WRN gene in mice accelerate mortality in a p53-null background. (57/2071)

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.  (+info)

The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa. (58/2071)

The opportunistic pathogen Pseudomonas aeruginosa uses intercellular signals to control the density-dependent expression of many virulence factors. The las and rhl quorum-sensing systems function, respectively, through the autoinducers N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone (C(4)-HSL), which are known to positively regulate the transcription of the elastase-encoding gene, lasB. Recently, we reported that a second type of intercellular signal is involved in lasB induction. This signal was identified as 2-heptyl-3-hydroxy-4-quinolone and designated the Pseudomonas quinolone signal (PQS). PQS was determined to be part of the quorum-sensing hierarchy since its production and bioactivity depended on the las and rhl quorum-sensing systems, respectively. In order to define the role of PQS in the P. aeruginosa quorum-sensing cascade, lacZ gene fusions were used to determine the effect of PQS on the transcription of the quorum-sensing system genes lasR, lasI, rhlR, and rhlI. We found that in P. aeruginosa, PQS caused a major induction of rhlI'-lacZ and had lesser effects on the transcription of lasR'-lacZ and rhlR'-lacZ. We also observed that the transcription of both rhlI'-lacZ and lasB'-lacZ was cooperatively effected by C(4)-HSL and PQS. Additionally, we present data indicating that PQS was not produced maximally until cultures reached the late stationary phase of growth. Taken together, our results imply that PQS acts as a link between the las and rhl quorum-sensing systems and that this signal is not involved in sensing cell density.  (+info)

Metallothionein, nitric oxide and zinc homeostasis in vascular endothelial cells. (59/2071)

Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N, N,N',N'-tetrakis(2. pyridylmethyl)ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic Ca(2+) via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall.  (+info)

Suppression of arterial intimal hyperplasia by cilostamide, a cyclic nucleotide phosphodiesterase 3 inhibitor, in a rat balloon double-injury model. (60/2071)

The effects of cilostamide, a cyclic nucleotide phosphodiesterase 3 (PDE3) selective inhibitor, on vascular intimal hyperplasia were evaluated using a single-balloon injury model and a double-injury model in which the rat common carotid artery was subjected to a second injury at a site injured 14 days previously. In the double-injury model, the second balloon injury caused more severe intimal hyperplasia (intima/media (IM) ratio, 1.88+/-0.10) than in the single-injury model (1.09+/-0.08). Histopathological study revealed that vascular smooth muscle cells (VSMC) were the predominant cell-type in the affected neointimal area. Oral administration of cilostamide for 2 weeks after the second injury suppressed intimal hyperplasia in the double-injury model (30 mg kg(-1) bid, 83% inhibition in terms of the IM ratio, P<0.05; 100 mg kg(-1) bid, 69% inhibition, P<0.05). Similar effects were also observed in the single-injury model with oral administration of cilostamide for 2 weeks (100 mg kg(-1) bid, 36% inhibition, P<0.01). Cilostamide inhibited DNA synthesis of cultured VSMC stimulated by foetal calf serum or different kinds of growth factors, but did not affect their migration stimulated by platelet-derived growth factor (PDGF)-BB. Cilostamide significantly increased the cyclic AMP concentration of VSMC dose-dependently. These results indicate that cilostamide suppresses intimal hyperplasia both in the single- and double-injury models of rat, presumably by inhibiting proliferation rather than migration of VSMC. It is suggested that PDE3 inhibitors might find application in preventing intimal hyperplasia following angioplasty such as percutaneous transluminal coronary angioplasty (PTCA) or stent.  (+info)

Quinolone resistance in enterotoxigenic Escherichia coli causing diarrhea in travelers to India in comparison with other geographical areas. (61/2071)

Enterotoxigenic Escherichia coli isolates were identified as a cause of traveler's diarrhea in 82 of 520 (16%) patients and tested for resistance to seven antimicrobial agents. Thirty patients (36%) needed antimicrobial therapy: 17 (56%) for persistence of symptoms and 13 (44%) for severity of symptoms. Ampicillin, tetracycline, and trimethoprim-sulfamethoxazole resistance was high. Chloramphenicol showed moderate activity, and amoxicillin plus clavulanic acid, nalidixic acid, and ciprofloxacin showed very good activity. Five nalidixic acid-resistant strains were isolated, four from patients visiting India.  (+info)

Microbiological transformation of enrofloxacin by the fungus Mucor ramannianus. (62/2071)

Enrofloxacin metabolism by Mucor ramannianus was investigated as a model for the biotransformation of veterinary fluoroquinolones. Cultures grown in sucrose-peptone broth were dosed with enrofloxacin. After 21 days, 22% of the enrofloxacin remained. Three metabolites were identified: enrofloxacin N-oxide (62% of the total absorbance), N-acetylciprofloxacin (8.0%), and desethylene-enrofloxacin (3.5%).  (+info)

8-Difluoromethoxy-4-quinolone derivatives as anti-feline immunodeficiency virus (FIV) agents: important structural features for inhibitory activity of FIV replication. (63/2071)

The inhibitory activities of various 8-difluoromethoxy-4-quinolone derivatives against feline immunodeficiency virus (FIV) replication in the chronically infected cell line P-CrFK were investigated. Certain derivatives were found to inhibit FIV production from P-CrFK cells in a dose-dependent manner without exhibiting cytotoxic effects at inhibitory concentrations. Based on this study, the structures important for anti-FIV activity are suggested to be (i) a carboxyl group at position C-3, and (ii) an aromatic modification at position 4 of the C-7 piperazinyl moiety.  (+info)

A molecular model of agonist and nonpeptide antagonist binding to the human V(1) vascular vasopressin receptor. (64/2071)

The affinity of the nonpeptide antagonist OPC-21268 is greater for the rat V(1) arginine vasopressin (AVP) receptor (V(1)R) than for the human V(1)R. Site-specific mutagenesis was carried out to identify the residues that determine interspecies selectivity for nonpeptide antagonist binding. The introduction of rat amino acids in position 224, 310, 324, or 337 of the human V(1)R sequence dramatically altered OPC-21268 affinity for the receptor, whereas binding of AVP, the peptide V(1)R antagonist d(CH(2))(5)Tyr(Me)AVP, and the nonpeptide V(1)R antagonist SR49059 was not altered by these mutations. Computer modeling explained the mutagenesis results. Docking of OPC-21268 onto a homology-built model of the V(1)R receptor yielded a model for the bound ligand in which the hydrophobic part is deeply embedded in the transmembrane region, whereas the polar part is located on the surface of the extracellular side. The increased affinity of the G337A mutant is due to two additional van der Waals contacts of the alanine methyl group with carbon atoms on the antagonist. The I310V mutant reduces the hydrophobicity in the vicinity of the polar oxygen atom of the antagonist. The I224V mutant relieves overcrowding in a hydrophobic binding pocket involving the aromatic residues Trp(175), Phe(179), Phe(307), and Trp(304). Finally, the E324D mutant enables the formation of a hydrogen bond of the carboxylate side chain with the amide side chain of Gln(311), which in turn forms a hydrogen bond with the N57 nitrogen atom of OPC-21268. Thus, a few residues, distinct from those involved in agonist binding, control interspecies selectivity toward OPC-21268 nonpeptide antagonist binding.  (+info)