Associations between air pollution in the industrial and suburban parts of Ostrava city and their use.

Selecting the locations and numbers of air quality monitoring stations is challenging as these are expensive to operate. Representative concentrations of pollutants in certain areas are usually determined by measuring. If there are significant correlations with concentrations of other pollutants or with other monitoring sites, however, concentrations could also be computed, partly reducing the costs. The aim of this study is to provide an overview of such possible relationships using data on concentrations of ambient air pollutants obtained in different areas of a larger city. Presented are associations between industrial (IP) and suburban parts (SP) as well as correlations between concentrations of various pollutants at the same site. Results of air pollutant monitoring come from Ostrava, an industrial city in Central Europe with a population of over 300,000. The study showed that certain pollutants were strongly correlated, especially particulate matter (r = 0.940) and ozone (r = 0.923) between the IP and SP. Statistically significant correlations were also found between different pollutants at the same site. The highest correlations were between PM10 and NO2 (r IP = 0.728; r SP = 0.734), NO2 and benzo(a)pyrene (r IP = 0.787; r SP = 0.697), and NO2 and ozone (r IP = -0.706; r SP = -0.686). This could contribute to more cost-effective solutions for air pollution monitoring in cities and their surroundings by using computational models based on the correlations, optimization of the network of monitoring stations, and the best selection of measuring devices.

In silico and experimental evaluation of DNA-based detection methods for the ability to discriminate almond from other Prunus spp.

Ten published DNA-based analytical methods aiming at detecting material of almond (Prunus dulcis) were in silico evaluated for potential cross-reactivity with other stone fruits (Prunus spp.), including peach, apricot, plum, cherry, sour cherry and Sargent cherry. For most assays, the analysis of nucleotide databases suggested none or insufficient discrimination of at least some stone fruits. On the other hand, the assay targeting non-specific lipid transfer protein (Röder et al., 2011, Anal Chim Acta 685:74-83) was sufficiently discriminative, judging from nucleotide alignments. Empirical evaluation was performed for three of the published methods, one modification of a commercial kit (SureFood allergen almond) and one attempted novel method targeting thaumatin-like protein gene. Samples of leaves and kernels were used in the experiments. The empirical results were favourable for the method from Röder et al. (2011) and a modification of SureFood allergen almond kit, both showing cross-reactivity <10(-3) compared to the model almond.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese.

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.

Evaluation of fungal and yeast diversity in Slovakian wine-related microbial communities.

Since the yeast flora of Slovakian enology has not previously been investigated by culture-independent methods, this approach was applied to two most common cultivars Frankovka (red wine) and Veltlin (white wine), and complemented by cultivation. Model samples included grapes, initial must, middle fermenting must and must in the end-fermentation phase. The cultured isolates were characterized by length polymorphism of rDNA spacer two region using fluorescence PCR and capillary electrophoresis (f-ITS PCR), and some were identified by sequencing. The microbial DNA extracted directly from the samples without cultivation was analysed by f-ITS PCR, amplicons were cloned and sequenced. The use of universal fungal primers led to detection of both yeasts and filamentous fungi. The amplicon of highest intensity and present in all the samples corresponded to Hanseniaspora uvarum. Other species demonstrated by both approaches included Saccharomyces sp., Metschnikowia pulcherrima or M. chrysoperlae, Candida zemplinina, Cladosporium cladosporioides, Botryotinia fuckeliana, Pichia anomala, Candida railenensis, Cryptococcus magnus, Metschnikowia viticola or Candida kofuensis, Pichia kluyveri or Pichia fermentas, Pichia membranifaciens, Aureobasidium pullulans, Alternaria alternata, Erysiphe necator, Rhodotorula glutinis, Issatchenkia terricola and Debaryomyces hansenii. Endemism of Slovakian enological yeasts was suggested on the level of minor genetic variations of the known species and probably not accounting for novel species. The prevalence of H. uvarum over Saccharomyces sp. in the samples was indicated. This is the first culture-independent study of Slovakian enology and the first time f-ITS PCR profiling was used on wine-related microbial communities.

A novel real-time polymerase chain reaction method for the detection of Brazil nuts in food.

A qualitative real-time PCR-based method for the detection of the Brazil nut (Bertholletia excelsa) component in food is described. The method consists of DNA isolation by chaotropic SPE and the subsequent PCR with Brazil nut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the gene encoding for the 11s globulin of the Brazil nut. The method was positive for eight Brazil nut samples from the market and negative for all other tested plant and meat materials used in the food industry (36 samples). The intrinsic LOD of the method was 10 pg Brazil nut DNA. Using a series of model nut paste mixtures with defined Brazil nut contents, a practical detection limit of 0.1% (w/w) Brazil nut was estimated. Practical applicability of the entire method was tested by qualitative analysis of nine food samples; no discrepancies between the declared and detected Brazil nut contents were found. The presented PCR-based method is useful for sensitive and selective detection of Brazil nut in food samples.

Molecular characterization of cytochrome P450 genes in the polycyclic aromatic hydrocarbon degrading Mycobacterium vanbaalenii PYR-1.

Mycobacterium vanbaalenii PYR-1 has the ability to degrade low- and high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). In addition to dioxygenases, cytochrome P450 monooxygenases have been implicated in PAH degradation. Three cytochrome P450 genes, cyp151 (pipA), cyp150, and cyp51, were detected and amplified by polymerase chain reaction from M. vanbaalenii PYR-1. The complete sequence of these genes was determined. The translated putative proteins were > or = 80% identical to other GenBank-listed mycobacterial CYP151, CYP150, and CYP51. Genes pipA and cyp150 were cloned, and the proteins partially expressed in Escherichia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at 450 nm in reduced carbon monoxide difference spectra. Monooxygenation metabolites of pyrene, dibenzothiophene, and 7-methylbenz[alpha]anthracene were detected in whole cell biotransformations, with E. coli expressing pipA or cyp150 when analyzed by gas chromatography/mass spectrometry. The cytochrome P450 inhibitor metyrapone strongly inhibited the S-oxidation of dibenzothiophene. Thirteen other Mycobacterium strains were screened for the presence of pipA, cyp150, and cyp51 genes, as well as the initial PAH dioxygenase (nidA and nidB). The results indicated that many of the Mycobacterium spp. surveyed contain both monooxygenases and dioxygenases to degrade PAHs. Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp. to metabolize polycyclic aromatic hydrocarbons.

Novel organization of genes in a phthalate degradation operon of Mycobacterium vanbaalenii PYR-1.

Mycobacterium vanbaalenii PYR-1 is capable of degrading polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites. This study identified and characterized a putative phthalate degradation operon in the M. vanbaalenii PYR-1 genome. A putative regulatory protein (phtR) was encoded divergently with five tandem genes: phthalate dioxygenase large subunit (phtAa), small subunit (phtAb), phthalate dihydrodiol dehydrogenase (phtB), phthalate dioxygenase ferredoxin subunit (phtAc) and phthalate dioxygenase ferredoxin reductase (phtAd). A 6.7 kb EcoRI fragment containing these genes was cloned into Escherichia coli and converted phthalate to 3,4-dihydroxyphthalate. Homologues to the operon region were detected in a number of PAH-degrading Mycobacterium spp. isolated from various geographical locations. The operon differs from those of other Gram-positive bacteria in both the placement and orientation of the regulatory gene. In addition, the M. vanbaalenii PYR-1 pht operon contains no decarboxylase gene and none was identified within a 37 kb region containing the operon. This study is the first report of a phthalate degradation operon in Mycobacterium spp.

Evidence for the existence of PAH-quinone reductase and catechol-O-methyltransferase in Mycobacterium vanbaalenii PYR-1.

Polycyclic aromatic hydrocarbon (PAH) quinone reductase (PQR) and catechol-O-methyltransferase (COMT), from the PAH-degrading Mycobacterium vanbaalenii PYR-1, were demonstrated to be constitutive enzymes located in the soluble fraction of cell extracts. PQR activities for the reduction of 9,10-phenanthrenequinone and 4,5-pyrene- quinone were 1.40+/-0.13 and 0.12+/-0.01 micromol min(-1) mg-protein(-1), respectively. The exogenous catechols alizarin, anthrarobin, 2,3-dihydroxynaphthalene and esculetin inhibited PQR activity. Anthrarobin (100 microM) and esculetin (100 microM) inhibited 4,5-pyrenequinone reduction by 64-92%. COMT was involved in the O-methylation of 1,2-dihydroxyphenanthrene to form 1-methoxy-2-hydroxyphenanthrene and 1,2-dimethoxyphenanthrene. Both pyrene and 1-hydroxypyrene were metabolized by M. vanbaalenii PYR-1 to form 1-methoxypyrene, 1-methoxy-2-hydroxypyrene, 1-hydroxy-2-methoxypyrene and 1,2-dimethoxypyrene. Among the catechols tested, anthrarobin showed the highest COMT activity (1.06+/-0.04 nmol/30 min(-1) mg-protein(-1)). These results suggest that the PQR and COMT activities of M. vanbaalenii PYR-1 may play an important role in the detoxification of PAH catechols.

Subcellular localization of yeast Sec14 homologues and their involvement in regulation of phospholipid turnover.

Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol-phosphatidylcholine transfer protein in vitro. In this work, the five homologues of Sec14p, Sfh1p-Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role. We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14. Using C-terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p. Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes. In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p-mediated phosphatidylcholine turnover. Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)-mediated turnover of phosphatidylcholine in vivo. In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p.

Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp.

Polycyclic aromatic hydrocarbon (PAH)-degrading genes nidA and nidB that encode the alpha and beta subunits of the aromatic ring-hydroxylating dioxygenase have been cloned and sequenced from Mycobacterium vanbaalenii PYR-1 [Khan et al., Appl. Environ Microbiol. 67 (2001) 3577-3585]. In this study, the presence of nidA and nidB in 12 other Mycobacterium or Rhodococcus strains was investigated. Initially, all strains were screened for their ability to degrade PAHs by a spray plate method, and for the presence of the dioxygenase Rieske center region by polymerase chain reaction (PCR). Only Mycobacterium sp. PAH 2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487) and M. frederiksbergense FAn9T (DSM 44346), all previously known PAH degraders, were positive in both tests. From the three positive strains, complete open reading frames of the nidA and nidB genes were amplified by PCR, using primers designed according to the known nidA and nidB sequences from PYR-1, cloned in the pBAD/Thio-TOPO vector and sequenced. The sequences showed >98% identity with the M. vanbaalenii PYR-1 nidA and nidB genes. Southern DNA-DNA hybridization using nidA and nidB probes from PYR-1 revealed that there is more than one copy of nidA and nidB genes in the strains PYR-1, BB1, PYR-GCK and FAn9T. However, only one copy of each gene was observed in PAH2.135.