Analysis of Black Fungal Biofilms Occurring at Domestic Water Taps (I): Compositional Analysis Using Tag-Encoded FLX Amplicon Pyrosequencing

Mass growth of dark fungal biofilms on water taps and associated habitats was observed in various German drinking water distribution systems recently. Customers of affected drinking water systems are anxious about potential and unknown health risks. These environments are known to harbour a fungal flora also comprising a variety of fungal opportunists that are well known to cause superficial mycoses in humans (Exophiala equina, Exophiala lecanii-corni) but are not known to establish dark biofilms so far. To gain profound insight on composition of respective biofilms, a metagenomic approach using Tag-Encoded FLX Amplicon Pyrosequencing (TEFAP) of the ribosomal internal transcribed spacer 2 region in comparison with a classical cultivation approach using Sabouraud agar with chloramphenicol and erythritol-chloramphenicol-agar was performed. E. lecanii-corni was found to be the major component in 10 of 13 biofilms analysed independently of the method used. Alternaria sp., E. equina, Fusarium spp. and Ochroconis spp. were also relatively abundant. As expected, TEFAP usually revealed a higher diversity than the cultivation approaches. For example, opportunistic species like Candida albicans or Exophiala dermatitidis were detected in very low amounts. In conclusion, TEFAP turned out to be a promising and powerful tool for the semi-quantitative analysis of fungal biofilms. Referring to relevant literature, potential biological hazards caused by fungi of the dark biofilms can be regarded as low.     

Reduction of Copper(II) in Fungal Laccase by Hydrated Electrons

FUNGAL laccase is a copper oxidase which catalyses the electron transfer from p-diphenols and related reductants to molecular oxygen¹. It contains four copper ions per molecule of protein which are bound to three distinctly different sites^1–4. Redox titrations show the involvement of four electron accepting sites in the reduction phase of the oxidase⁵. Kinetic studies by stopped flow and relaxation methods have suggested that the Cu(II) ion designated type 1, with an intense absorption band at 610 nm and very small hyperfine splitting in its electron spin resonance signal, is the first site to accept the incoming electrons (ref. 6 and I. P., results to be published). From type 1 Cu(II) the electron is probably transferred to the other sites by an intramolecular process.     

Erratum to: The Analysis of Cu(II)/Zn(II) Cyclopeptide System as Potential Cu,ZnSOD Mimic Center

International Journal of Peptide Research and Therapeutics -     

The Analysis of Cu(II)/Zn(II) Cyclopeptide System as Potential Cu,ZnSOD Mimic Center

In this paper are presented the features of copper (II) and zinc (II) heteronuclear complexes of the cyclic peptide—c(HKHGPG)₂. The coordination properties of ligand were studied by potentiometric, UV–Vis and CD spectroscopic methods. These experiments were carried out in aqueous solutions at 298 K depending on pH. It turned out that in a physiological pH dominates Cu(II)/Zn(II) complex ([CuZnL]⁴⁺) which could mimic the active center of superoxide dismutase (Cu,ZnSOD). In next step we performed in vitro research on Cu,ZnSOD activity for [CuZnL]⁴⁺ complex existing in 7.4 pH by the method of reduction of nitroblue tetrazolium (NBT). Also mono- and di-nuclear copper (II) complexes of this ligand were examined. The ability of inhibition free radical reaction were compared for all complexes. The results of these studies show that Cu(II) mono-, di-nuclear and Cu(II)/Zn(II) complexes becoming to new promising synthetic superoxide dismutase mimetics, and should be considered for further biological assays.     

Water Relations of White Clover (Trifolium repens): Water Potential Gradients and Plant Morphology

Water potential, osmotic potential, pressure potential and relativewater content were measured in stolons and leaves of white cloverplants grown under a range of conditions of water supply andevaporative demand. The importance of adventitious roots fromthe nodes was examined. Gradients along stolons were alwaysextremely small, of the order of only 01 MPa. Stolon up waterpotential was representative of plant water status regardlessof stolon length, presence/absence of nodal roots, degree ofwater stress and evaporative demand. It is concluded that waterconduction along stolons was very good. Gradients were foundto exist along petioles; they may have a greater resistanceto water flow than stolons. The relationship between water fluxand stem anatomy, and the importance of differential flow ratesthrough stolons and petioles to plant behaviour during waterstress, are discussed. Trifolium repens L., white clover, water relations     

Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: II. Regulation by Nitrate Flux at Low Leaf Water Potential

Experiments were conducted to determine whether the nitrate flux to the leaves or the nitrate content of the leaves regulated the nitrate reductase activity (NRA) in leaves of intact maize (Zea mays L.) seedlings having low water potentials (ψ_w) when other environmental and endogenous factors were constant. In seedlings that were desiccated slowly, the nitrate flux, leaf nitrate content, and NRA decreased as ψ_w decreased. The decrease in nitrate flux was caused by a decrease in both the rate of transpiration and the rate of nitrate delivery to the transpiration stream. Upon rewatering, the recovery in NRA was correlated with the nitrate flux but not the leaf nitrate content.     

Water soluble platinum(II) and palladium(II) complexes of alkyl sulfonated phosphines

The reactions of the alkylsulfonated phosphines LM=Ph₂P(CH₂)_nSO₃Na/K (n=2, 3, 4) with K₂PtCl₄ and K₂PdCl₄ have been studied in homogeneous aqueous solution as a function of pH. In homogeneous acidic solution the protonated phosphines react to give cis- and trans-PtCl₂(LH)₂. The biphasic reaction between 1,5-cyclooctadiene platinum(II) chloride in dichloromethane and acidified aqueous LNa/K gives a higher proportion of the cis isomer. In neutral solution the initial reaction to give [PtCl(LNa/K)₃]⁺Cl⁻ is followed by slow formation of cis-PtCl₂(LNa/K)₂. K₂PdCl₄ reacts more rapidly to give PdCl₂(LNa/K)₂. In homogeneous alkaline solution rapid oxidation of the phosphine occurs with only small amounts of platinum complex being observable. The biphasic reaction yields phosphine oxide in the aqueous layer and a small amount of the chelate complexes PtL₂ in the organic. Representative complexes have been isolated and characterised and the mechanisms for the reactions discussed. The electrospray mass spectra of solutions of the isolated complexes have been recorded in both positive and negative ionisation modes. The positive ionisation spectra are complicated, but platinum and palladium containing ions derived from loss of chloride, H⁺ and HCl are observed in the negative ionisation spectra.     

Seasonal variability of phytoplankton at Varna Bay (Black Sea)

Intrinsic fluorescence and SDS-PAGE analysis were employed to study the seasonal qualitative and quantitative changes of phytoplankton composition at Varna Bay (Black Sea). Variation in the maximum emission wavelength (lambda(max)) of the phytoplankton proteins (398 nm in the summer and 340 nm in the spring) was observed. In addition, a decrease in fluorescence intensity, and a shift in lambda(max) as a result of changes in phytoplankton protein stability, according to the season, was noted. Similarly, SDS-PAGE analysis showed different protein patterns for each season, for example in summertime the major protein constituents were of 14, 37, 48 and 70 kDa, while in the springtime the sizes ranged between 38 and 48 kDa. In general, higher carbohydrate and protein contents correlated with larger phytoplankton biomass found during the summer. The dominant species, the Bacillariophyceae and Dinophyceae, were found to be present in the water body in an alternate pattern. All of these changes could be accounted for by the adaptation of the organism to seasonal variations that modify the sea environment at Varna Bay.     

Domestic Birds as Source of Cryptococcus deuterogattii (AFLP6/VGII): Potential Risk for Cryptococcosis

Mycopathologia - Cryptococcosis is an infection caused by encapsulated basidiomycetous yeasts belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. It is acquired through...     

Fungal Parasites of the Phytoplankton. II (Studies on British Chytrids, XII)

This paper brings together the species of Chytrid fungi recordedfrom large bodies of water in the British Isles. Four new speciesare described.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Potential methods for selective accumulation of nickel(II) ions by plants

The selective uptake of nickel ions by certain plant species is described. The observed selectivity relative to metal ions such as cobalt and iron cannot be explained on the known binding constants to oxygen-donor ligands, e.g., citrate. Nitrogen donors are shown to have adequate selective interaction.     

Key to Species of Aulacaspis (Hemiptera: Diaspididae) Intercepted at the Republic of Korea Ports of Entry

Six species of aulacaspis scales, Aulacaspis crawii (Cockerell), Aulacaspis madiunensis (Zehntner), Aulacaspis neospinosa Tang, Aulacaspis spinosa (Maskell), Aulacaspis tubercularis Newstead and Aulacaspis yasumatsui Takagi, have been taken in quarantine at the republic of Korea ports of entry between 1996 and 2005. We have also found the specimens of A. yasumatsui Takagi on Cycas plants from Malaysia and Taiwan and provide a key with illustrative photographs to identify intercepted specimens of Aulacaspis.     

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the x,y and x,z planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.     

Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline,Theobromine and Caffeine) Binding with DNA

Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg²⁺ and during helix-coil transitions of DNA by temperature (T_m) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10³ M⁻¹, DNA-theobromine = 1.1×10³ M⁻¹, and DNA-Caffeine = 3.8×10³ M⁻¹. On the other hand T_m/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg²⁺, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg²⁺. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.     

Synthesis and properties of trinuclear polypyridyl complexes Ru(II)-Co(II)-Ru(II) and Ru(II)-Co(III)-Ru(II): Their photoinduced interconversion

The trinuclear [{Ru^II(bpy)₂(bpy-terpy)}₂Co^II]⁶⁺ complex (1⁶⁺) in which a Co(II)-bis-terpyridine-like centre is covalently linked to two Ru(II)-tris-bipyridine-like moieties by a bridging bipyridine-terpyridine ligand has been synthesised and characterised. Its electrochemical, photophysical and photochemical properties have been investigated in CH₃CN. The cyclic voltammetry exhibits two successive reversible oxidation processes, corresponding to the Co^III/Co^II and Ru^III/Ru^II redox couples at E_1/2 = −0.06 and 0.91 V vs Ag/Ag⁺ 10 mM, respectively. The one-electron oxidized form of the complex, [{Ru^II(bpy)₂(bpy-terpy)}₂Co^III]⁷⁺ (1⁷⁺) obtained after exhaustive electrolysis carried out at 0.2 V is fully stable. 1⁶⁺ and 1⁷⁺ are only poorly luminescent, indicating that the covalent linkage of the Ru(II)-tris-bipyridine centre to the cobalt subunit leads to a strong quenching of the Ru^II excited state by an intramolecular process. Luminescence lifetime experiments carried out at different temperatures indicate that the transfer is more efficient for 1⁷⁺ compare to 1⁶⁺ due to lower activation energy. Continuous irradiation of 1⁷⁺ performed at 405 nm in the presence of P(Ph)₃ acting as sacrificial electron donor leads to its quantitative reduction into 1⁶⁺, whereas similar experiment starting from 1⁶⁺ with a sulfonium salt as sacrificial electron acceptor converts 1⁶⁺ into 1⁷⁺ with a slower rate and a maximum yield of 80%. These photoinduced electron transfers were followed by UV-Visible spectroscopy and compared with those obtained with a simple mixture of both mononuclear parent complexes i.e. [Ru^II(bpy)₃]²⁺ and [Co^II(tolyl-terpy)₂]²⁺ or [Co^III(tolyl-terpy)₂]³⁺ (tolyl-terpy = 4′-(4-methylphenyl)-2,2′:6′,2′′-terpyridine).     

Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.     

Resistance to Water Transport in Shoots of Vitis vinifera L. : Relation to Growth at Low Water Potential

Apparent resistances to water transport in the liquid phase were determined from measurements of soil, root, basal shoot internode, shoot apex, and leaf water potentials and water flux in Vitis vinifera (cv White Riesling) during soil drying. Predawn water potential differences (ΔΨ) in the shoots accounted for 20% of the total ΔΨ between the soil and the shoot apex when plants were well-watered but increased to about 90% when shoot growth ceased. The ΔΨ from soil to root was essentially constant during this period. At low water potential, the ΔΨ in the shoot was persistent when transpiration was low (predawn) or completely prevented (plant bagging). The apparent hydraulic resistance between the basal shoot internode and most rapidly expanding leaf (or shoot apex) increased several-fold when water was withheld. Leaf and internode expansion both exhibited high sensitivity to increasing hydraulic resistance. Measurements of pneumatic resistance to air flow through frozen internode segments indicated progressive vapor-filling of vessels as soil drying progressed. From these observations and others in the literature, it was suggested that embolization may be a common occurrence and play an important role in the inhibition of shoot growth at moderate water deficits.     

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.