High Prevalence of Vancomycin-Resistant Enterococci in Swedish Sewage

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden (n = 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the vanA gene, and the majority (24 of 35) of the isolates were Enterococcus faecium. Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.     

Temperature-dependent structural transition following X-ray-induced metal center reduction in oxidized cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in the inner membrane of mitochondria. It contains four metal redox centers, two of which, Cu_B and heme a₃, form the binuclear center (BNC), where dioxygen is reduced to water. Crystal structures of CcO in various forms have been reported, from which ligand-binding states of the BNC and conformations of the protein matrix surrounding it have been deduced to elucidate the mechanism by which the oxygen reduction chemistry is coupled to proton translocation. However, metal centers in proteins can be susceptible to X-ray-induced radiation damage, raising questions about the reliability of conclusions drawn from these studies. Here, we used microspectroscopy-coupled X-ray crystallography to interrogate how the structural integrity of bovine CcO in the fully oxidized state (O) is modulated by synchrotron radiation. Spectroscopic data showed that, upon X-ray exposure, O was converted to a hybrid O∗ state where all the four metal centers were reduced, but the protein matrix was trapped in the genuine O conformation and the ligands in the BNC remained intact. Annealing the O∗ crystal above the glass transition temperature induced relaxation of the O∗ structure to a new R∗ structure, wherein the protein matrix converted to the fully reduced R conformation with the exception of helix X, which partly remained in the O conformation because of incomplete dissociation of the ligands from the BNC. We conclude from these data that reevaluation of reported CcO structures obtained with synchrotron light sources is merited.     

Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.     

Interpretation of the seasonal variations of nutrients in a mediterranean lagoon: étang de Thau

A network of 63 stations was used on four occasions (June 1986, October 1986, February 1987, and May 1987) to study the spatio-temporal distribution of inorganic nutrients in Thau Lagoon (l'étang de Thau), which covers 7500 hectares on the French Mediterranean coast. Three environmental factors, revealed by multiple regression models, govern the distributions observed. Allochthonous inputs from the watershed enrich the environment with nitrogen and phosphorus compounds in the winter and autumn. Internal sources are essentially localized in the shellfish breeding zone of the lagoon. In the summer, shellfish excretions and the rapid remineralization of organic deposits produce ammonium ions. Uptake by phytoplankton has a much larger impact on the seasonal variation of inorganic nitrogen than on that of phosphorus; the latter is present in excess in the lagoon waters. Thus, nitrogen appears to be the primary limiting nutrient for the development of the chorophyllous biomass.     

Protein methylation in animal cells. I. Purification and properties of S-adenosyl-L-methionine:protein (arginine) N-methyltransferase from Krebs II ascites cells.

1. A protein methylase which specifically transfers methyl groups from S-adenosyl-L-methionine to arginine residues of histones has been substantially purified from Krebs II ascites cells. The purified enzyme was obtained free of contamination by other protein methyl transferases specific for carboxyl and lysine residues. This latter activity copurified with the present enzyme until advanced stages of purification. 2. The purified enzyme does not require any divalent cation for maximum activity. It is inhibited by ionic strength, N-ethylmaleimide and S-adenosyl-L-homocysteine. It has an apparent molecular weight on gel filtration of approx. 5 . 10(5). A Km value for S-adenosyl-L-methionine of 2.5 . 10(-6) M was determined, while the dissociation constant Ki for S-adenosyl-L-homocysteine, which acts as a competitor, was 1.4 . 10(-6) M.     

Cardiorenal abnormalities associated with high sodium intake: correction by spironolactone in rats

Reversal by the mineralocorticoid receptor antagonist spironolactone on cardiac and renal abnormalities, associated with long-term (since weaning) administration of a high (2 and 8% NaCl chow, HS2 and HS8) sodium diet, was assessed in Sprague-Dawley rats. At the age of 5 mo, spironolactone (20 or 100 mg/kg, gavage) or placebo were given for 14 days to HS2 and HS8 rats. A group fed a regular diet (0.8% NaCl, NS) remained untreated. High sodium intake had no detectable effect on blood pressure; however, cardiac mass index and cross-sectional area of the carotid artery, as well as albuminuria, were increased only in the HS8 group compared with the control group on NS diet. In addition, a marked reduction in glomerular filtration rate (by 40%), associated with a nonproportional fall in renal plasma flow (thus resulting in a decrease in filtration fraction), was observed only in the HS8 group. No change in cardiac and renal fibrosis was detected. Production of the reactive oxygen species (ROS) by aortic tissue was increased in HS8 rats, whereas ROS production by the heart was unaffected. Only the high dose of spironolactone was effective, as it markedly reversed the cardiac hypertrophy and renal hypofiltration associated with the HS8 feeding. The changes were observed in the absence of any effect on systemic blood pressure and production of ROS. These observations favor aldosterone's role in the deleterious effects of marked and prolonged increases in sodium intake.     

Cell proliferation and gill morphology in anoxic crucian carp

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.     

中药王不留行化学成分的研究

从河北产麦蓝菜(Vaccaria segetalis(Neck)Garcke.)的种子王不留行中分离得到5个化合物,经波谱分析和化学方法分别鉴定为:Tetracosanoic acid(1),E-3-phenyl-2-Propenoic acid(2),stigmast-7,22-dien-3-ol(3),stigmast-7,22-dien-3-ol 3β-O-[β-D-glucopy-ranoside](4),9,12-octadecadienoic acid N-(2-hydroxyethyl)(5)。以上各化合物都是首次从该植物中分得。化合物5为首次从天然界分得。     

Mechanism and Factors Influencing the Veronal Inhibition of Bacterial Spore Germination

The inhibitory effect of sodium 5,5-diethyl barbiturate (Veronal) on the L-alanine-induced initiation of germination of Bacillus subtilis spores was examined. Veronal reversibly inhibited the initiation of germination by a noncompetitive mechanism. The inhibition was time-independent and it took place whether L-alanine was or was not allowed to permeate the spore before the addition of the inhibitor. The concentration of the inhibitor and the pH of the initiation system were important factors determining the effectiveness of Veronal as an inhibitor. The magnitude of the inhibition increased linearly with decreasing pH at constant concentration and with increasing concentration at constant pH. These results suggest that the inhibition involves a permeability phenomenon related to the access of drug to the active sites in the spore and that the entry of Veronal into the spores is regulated by the concentration of undissociated molecule. At the physiologically important pH of 7.4, initiation with alanine in phosphate buffer at high spore densities (about 10(9) spores per ml) was 50% inhibited by 4 mM Veronal, and 8mM Veronal inhibited initiation completely. L-Alanine initiation in tris(hydroxymethyl)amino-methane-hydrochloride buffer was completely inhibited by 5 mM Veronal. The inhibition could be partially reversed by the combined addition of D-fructose, D-glucose, and K(+). Possible reasons for the failure of otherwise inhibitory concentrations of Veronal to inhibit completely the L-alanine-induced initiation when a combination of fructose, glucose, and K(+) was present and a suggested relationship to two functional roles of L-alanine in the initiation of germination are discussed.