Zinc(II) tweezers containing artificial peptides mimicking the active site of phosphotriesterase: The catalyzed hydrolysis of the toxic organophosphate parathion

Two new ligand-containing histidine based on N,N′,N″-tris(N-benzyl-l-histidinyl)tri(2-aminoethyl)amine, L¹, namely N,N′,N″-tris[(1S)-2-methoxy-2-oxy-1-(1-benzylimidazol-4-ylmethyl)]nitrilotriacetamide L² and N,N′,N″-tris{N-benzyl-N-[N-benzyl-N-(N-benzyl-l-histidinyl)-l-histidinyl]-l-histidinyl}tri(2-aminoethyl)amine L³ were prepared. Zinc(II) binding studies by these ligand systems were analyzed by means of potentiometric and ¹H NMR titrations in aqueous methanol (33 % v/v). Subsequently their zinc(II) complexes [L¹Zn(H₂O)](ClO₄)₂·HClO₄ (1), [L²Zn(OH₂)](ClO₄)₂·H₂O (2), and ([L³Zn₃(H₂O)₃](ClO₄)₆·3HClO₄·5H₂O (3), respectively were synthesized and characterized. The reactivity of the trinuclear complex (3) toward the hydrolysis of the toxic organophosphate parathion was investigated and compared with that of the mononuclear reference complex (1). From the pH dependence of the apparent rate constants, and the deprotonation constant (pK_a) of the coordinated water molecules in (1), the active species were confirmed to be {[HL¹Zn(OH)]²⁺/[L¹Zn(H₂O)]²⁺} at pH 8.5. The trizinc complex (3) effects hydrolysis of parathion, with three times rate enhancement over the mononuclear (1), indicating that cooperative action of the three zinc centers is limited.     

Environmental changes affect ecosystem services of the intermittent Lake Cerknica

Lake Cerknica is an intermittent wetland with seasonal water level fluctuations. This paper discusses the possible changes of ecosystem services due to altered ecosystem functions caused by regional climate change. For this purpose, the lake's water regime was analysed and biomass production of common reed (Phragmites australis), the prevailing species, was related to ambient temperatures and water level. The effect of the latter was also examined for reed transpiration rate and plant diversity. The results revealed a gradual loss of seasonality of floods and droughts. High water level in winter months and temperatures at the beginning as well as the end of vegetation period were found to have a significant influence on reed biomass. The plant diversity research in six subsequent years in three different aquatic habitats revealed trends of a decrease due to increased water depth during the vegetation period. The complexity of the system and consequently ecosystem services might also be affected due to changes of other processes, which revealed to be related to water level; namely transpiration rate, plant mycorrhizal colonisation and soil mineralisation.     

Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.     

A comparative genomic analysis of the calcium signaling machinery in Neurospora crassa, Magnaporthe grisea, and Saccharomyces cerevisiae

A large number of Ca2+ -signaling proteins have been previously identified and characterized in Saccharomyces cerevisiae but relatively few have been discovered in filamentous fungi. In this study, a detailed, comparative genomic analysis of Ca2+ -signaling proteins in Neurospora crassa, Magnaporthe grisea, and S. cerevisiae has been made. Our BLAST analysis identified 48, 42, and 40 Ca2+ -signaling proteins in N. crassa, M. grisea, and S. cerevisiae, respectively. In N. crassa, M. grisea, and S. cerevisiae, 79, 100, and 13% of these proteins, respectively, were previously unknown. For N. crassa, M. grisea, and S. cerevisiae, respectively, we have identified: three Ca2+ -permeable channels in each species; 9, 12, and 5 Ca2+/cation-ATPases; eight, six, and four Ca2+ -exchangers; four, four, and two phospholipase C's; one calmodulin in each species; and 23, 21, and 29 Ca2+/calmodulin-regulated proteins. Homologs of a number of key proteins involved in the release of Ca2+ from intracellular stores, and in the sensing of extracellular Ca2+, in animal and plant cells, were not identified. The greater complexity of the Ca2+ -signaling machinery in N. crassa and M. grisea over that in S. cerevisiae probably reflects their more complex cellular organization and behavior, and the greater range of external signals which filamentous fungi have to respond to in their natural habitats. To complement the data presented in this paper, a comprehensive web-based database resource (http://www.fungalcell.org/fdf/) of all Ca2+ -signaling proteins identified in N. crassa, M. grisea, and S. cerevisiae has been provided.     

The quality of the aquatic environment and macrophytes of karstic watercourses

The quality of the aquatic environment and macrophytes were surveyed in the case of the remarkable karstic river Ljubljanica s. lat. (Slovenia). It consists of seven intermittent and permanent surface watercourses, connected by groundwater flows. We assessed 11 parameters describing the land use beyond the riparian zone, the structure of the riparian zone and stream channel morphology, as well as the distribution and abundance of macrophytes. The intermittent watercourses exhibited a relatively high level of naturalness, because the variable water regime prevents intensive human activity. Greater influence of the human activity on the permanent watercourses resulted in a modified riverine environment. The share of different ecological groups of macrophytes, i.e. submerged, emerged and amphibious species, reflected the water regimes of the different watercourses. A total of 62 taxa of macrophytes were identified in about 100 km of length examined. Of these, amphibious macrophytes dominated as a consequence of intermittent water regime. The relationship between environmental parameters and macrophytes, and the effects of the intermittent hydrology were investigated. All environmental parameters explained 28% of the variance in the distribution of macrophytes in watercourses, as determined by canonical correspondence analysis. Completeness of the riparian zone, bank undercutting and sediment accumulation were found to be the most influential.     

Investigation of antibiotic and antibacterial agent cross-resistance in target bacteria from homes of antibacterial product users and nonusers

AIM: To describe the relationship between antibiotic and antibacterial resistance in environmental and clinical bacteria from home environments across geographical locations, relative to the use or nonuse of antibacterial products, with a focus on target organisms recognized as potential human pathogens. METHODS AND RESULTS: In a randomized study, environmental and clinical samples were collected from the homes of antibacterial product users (n=30) and nonusers (n=30) for the isolation of target bacteria for antibiotic and antibacterial testing in three geographical areas (in USA and UK). Isolates were tested for antibiotic susceptibility, with selected antibiotic-resistant and antibiotic-susceptible isolates tested against four common antibacterial agents (triclosan, para-chloro-meta-xylenol, pine oil and quaternary ammonium compound). Prequalified users and nonusers at each location were randomly selected after meeting exclusionary criteria. Of 1238 isolates, more target bacteria were recovered from nonuser than user homes. Of Staphylococcus aureus isolates (n=33), none showed resistance to oxacillin or vancomycin; for Enterococcus sp. (n=149), none were resistant to ampicillin or vancomycin; and for Klebsiella pneumoniae (n=54)and Escherichia coli (n=24), none were resistant to third generation cephalosporins. Antibiotic resistance to one or more of the standard test panel drugs for Gram-positive and Gram-negative target bacteria was comparable between nonuser and user homes for both environmental and clinical isolates [e.g. resistance of environmental coagulase-negative (CN) Staphylococcus sp. was 73.8% (124/168) from nonuser homes and 73.0% (111/152) from user homes, and Enterobacteriaceae other than E. coli, 75.9% (186/245) from nonuser homes compared with 78.0% from user homes]. Of 524 Gram-negatives tested against preferred/alternative drugs, 97.1% (509/524) were susceptible to all antibiotics, across both groups. Isolates of S. aureus, Enterococcus sp. and CN Staphylococcus sp. susceptible to all preferred treatment drugs showed comparable antibacterial minimum inhibitory concentration (MIC) results between nonuser and user home isolates. For Gram-positives resistant to one or more preferred drugs, greatest resistance to antibacterial active ingredients was found in the nonuser group. For Gram-negatives, the antibacterial MIC data were comparable for isolates that were fully susceptible and resistant to one or more preferred/alternative treatment antibiotics. CONCLUSIONS: The results showed a lack of antibiotic and antibacterial agent cross-resistance in target bacteria from the homes of antibacterial product users and nonusers, as well as increased prevalence of potential pathogens in nonuser homes. SIGNIFICANCE AND IMPACT OF THE STUDY: It refutes widely publicized, yet unsupported, hypotheses that use of antibacterial products facilitates the development of antibiotic resistance in bacteria from the home environment.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. III. Isolation and Genetic Properties of Group III Suppressors

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. Previous evidence indicated that suppressors of one group (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) represent mutations in the structural genes for glycyl-tRNA's. Suppressors of the other group (Group III: SUF2 and SUF7) were less well characterized. Although they suppressed some ICR-revertible mutations, they failed to suppress Group II frameshift mutations. This communication provides a more thorough characterization of the Group III suppressors and describes the isolation and properties of four new suppressors in that group (SUF8, SUF9, SUF10 and suf11).——In our original study, Group III suppressors were isolated as revertants of the Group III mutations his4–712 and his4–713. All suppressors obtained as ICR-induced revertants of these mutations mapped at the SUF2 locus near the centromere of chromosome III. Suppressors mapping at other loci were obtained in this study by analyzing spontaneous and UV-induced revertants of the Group III mutations. SUF2 and SUF10 suppress both Group III his4 mutations, whereas SUF7, SUF8, SUF9 and suf11 suppress his4–713, but not his4–712. All of the suppressors except suf11 are dominant in diploids homozygous for his4-713. The suppressors fail to suppress representative UAA, UAG and UGA nonsense mutations.——SUF9 is linked to the centromere of chromosome VI, and SUF10 is linked to the centromere of chromosome XIV. A triploid mapping procedure was used to determine the chromosome locations of SUF7 and SUF8. Subsequent standard crosses revealed linkage of SUF7 to cdc5 on chromosome XIII and linkage of SUF8 to cdc12 and pet3 on chromosome VIII.