Short communication: Changes under low ambient temperatures in the milk lipodome and metabolome of mid-lactation cows after dehorning as a calf.

Horns are living tissue and cows can use their horns for thermoregulatory purposes. We investigated the effect of the presence of horns on the metabolome of milk serum and lipidome of milk fat, to assess the physiological effect of dehorning. Milk sampling took place at low ambient temperatures of -6 to 2°C. Horned and dehorned cows were kept in a mixed herd of Holstein Friesian and Brown Swiss cows. The hypothesis was that horned cows needed to increase their metabolism to compensate for additional heat loss through the presence of their horns. No differences were observed in milk yield, milk solids, and somatic cell counts between horned and dehorned cows. For the milk metabolome, horned cows showed an upregulation of several glucogenic AA that could be transformed into glucose for energy supply and a downregulation of sugar intermediates and γ-glutamylcysteine compared with dehorned cows. The fatty acid (FA) composition in horned cows showed a shift toward decreased odd medium-chain FA (C7:0, C9:0, and C11:0) and increased cis-vaccenic acid (C18:1n-7 cis-11) and stearidonic acid (C18:4n-3). The changes in milk composition related to additional heat loss in horned cows indicate a competition in C3 metabolism for glucose synthesis and de novo FA synthesis under cold stress.

Bioavailability and uptake of smelter emissions in freshwater zooplankton in northeastern Washington, USA lakes using Pb isotope analysis and trace metal concentrations.

The upper Columbia River and associated valley systems are highly contaminated with metal wastes from nearby smelting operations in Trail, British Columbia, Canada (Teck smelter), and to a lesser extent, Northport, Washington, USA (Le Roi smelter). Previous studies have investigated depositional patterns of airborne emissions from these smelters, and documented the Teck smelter as the primary metal contamination source. However, there is limited research directed at whether these contaminants are bioavailable to aquatic organisms. This study investigates whether smelter derived contaminants are bioavailable to freshwater zooplankton. Trace metal (Zn, Cd, As, Sb, Pb and Hg) concentrations and Pb isotope compositions of zooplankton and sediment were measured in lakes ranging from 17 to 144 km downwind of the Teck smelter. Pb isotopic compositions of historic ores used by both smelters are uniquely less radiogenic than local geologic formations, so when zooplankton assimilate substantial amounts of smelter derived metals their compositions deviate from local baseline compositions toward ore compositions. Sediment metal concentrations and Pb isotope compositions in sediment follow significant (p < 0.001) negative exponential and sigmoidal patterns, respectively, as distance from the Teck smelting operation increases. Zooplankton As, Cd, and Sb contents were related to distance from the Teck smelter (p < 0.05), and zooplankton Pb isotope compositions suggest As, Cd, Sb and Pb from historic and current smelter emissions are biologically available to zooplankton. Zooplankton from lakes within 86 km of the Teck facility display isotopic evidence that legacy ore pollution is biologically available for assimilation. However, without water column data our study is unable to determine if legacy contaminants are remobilized from lake sediments, or erosional pathways from the watershed.

Effect of outpatient antibiotics for urinary tract infections on antimicrobial resistance among commensal Enterobacteriaceae: a multinational prospective cohort study.

OBJECTIVES: We quantified the impact of antibiotics prescribed in primary care for urinary tract infections (UTIs) on intestinal colonization by ciprofloxacin-resistant (CIP-RE) and extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE), while accounting for household clustering. METHODS: Prospective cohort study from January 2011 to August 2013 at primary care sites in Belgium, Poland and Switzerland. We recruited outpatients requiring antibiotics for suspected UTIs or asymptomatic bacteriuria (exposed patients), outpatients not requiring antibiotics (non-exposed patients), and one to three household contacts for each patient. Faecal samples were tested for CIP-RE, ESBL-PE, nitrofurantoin-resistant Enterobacteriaceae (NIT-RE) and any Enterobacteriaceae at baseline (S1), end of antibiotics (S2) and 28 days after S2 (S3). RESULTS: We included 300 households (205 exposed, 95 non-exposed) with 716 participants. Most exposed patients received nitrofurans (86; 42%) or fluoroquinolones (76; 37%). CIP-RE were identified in 16% (328/2033) of samples from 202 (28%) participants. Fluoroquinolone treatment caused transient suppression of Enterobacteriaceae (S2) and subsequent two-fold increase in CIP-RE prevalence at S3 (adjusted prevalence ratio (aPR) 2.0, 95% CI 1.2-3.4), with corresponding number-needed-to-harm of 12. Nitrofurans had no impact on CIP-RE (aPR 1.0, 95% CI 0.5-1.8) or NIT-RE. ESBL-PE were identified in 5% (107/2058) of samples from 71 (10%) participants, with colonization not associated with antibiotic exposure. Household exposure to CIP-RE or ESBL-PE was associated with increased individual risk of colonization: aPR 1.8 (95% CI 1.3-2.5) and 3.4 (95% CI 1.3-9.0), respectively. CONCLUSIONS: These findings support avoidance of fluoroquinolones for first-line UTI therapy in primary care, and suggest potential for interventions that interrupt household circulation of resistant Enterobacteriaceae.

Mixed colonies of Aspergillus niger and Aspergillus oryzae cooperatively degrading wheat bran.

In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions are often described as antagonistic, but can also be neutral or cooperative, and are generally associated with a metabolic change of each partner and cause a change in the pattern of produced bioactive molecules. A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). The co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture. Microscopic observation correlated with quantitative PCR and proteomic data suggest that the XlnR Knock-out strain benefit from the release of sugars by the wild type strain to support its growth.

Milk metabolome relates enteric methane emission to milk synthesis and energy metabolism pathways.

Methane (CH4) emission of dairy cows contributes significantly to the carbon footprint of the dairy chain; therefore, a better understanding of CH4 formation is urgently needed. The present study explored the milk metabolome by gas chromatography-mass spectrometry (milk volatile metabolites) and nuclear magnetic resonance (milk nonvolatile metabolites) to better understand the biological pathways involved in CH4 emission in dairy cattle. Data were used from a randomized block design experiment with 32 multiparous Holstein-Friesian cows and 4 diets. All diets had a roughage:concentrate ratio of 80:20 (dry matter basis) and the roughage was grass silage (GS), corn silage (CS), or a mixture of both (67% GS, 33% CS; 33% GS, 67% CS). Methane emission was measured in climate respiration chambers and expressed as CH4 yield (per unit of dry matter intake) and CH4 intensity (per unit of fat- and protein-corrected milk; FPCM). No volatile or nonvolatile metabolite was positively related to CH4 yield, and acetone (measured as a volatile and as a nonvolatile metabolite) was negatively related to CH4 yield. The volatile metabolites 1-heptanol-decanol, 3-nonanone, ethanol, and tetrahydrofuran were positively related to CH4 intensity. None of the volatile metabolites was negatively related to CH4 intensity. The nonvolatile metabolites acetoacetate, creatinine, ethanol, formate, methylmalonate, and N-acetylsugar A were positively related to CH4 intensity, and uridine diphosphate (UDP)-hexose B and citrate were negatively related to CH4 intensity. Several volatile and nonvolatile metabolites that were correlated with CH4 intensity also were correlated with FPCM and not significantly related to CH4 intensity anymore when FPCM was included as covariate. This suggests that changes in these milk metabolites may be related to changes in milk yield or metabolic processes involved in milk synthesis. The UDP-hexose B was correlated with FPCM, whereas citrate was not. Both metabolites were still related to CH4 intensity when FPCM was included as covariate. The UDP-hexose B is an intermediate of lactose metabolism, and citrate is an important intermediate of Krebs cycle-related energy processes. Therefore, the negative correlation of UDP-hexose B and citrate with CH4 intensity may reflect a decrease in metabolic activity in the mammary gland. Our results suggest that an integrative approach including milk yield and composition, and dietary and animal traits will help to explain the biological metabolism of dairy cows in relation to methane CH4 emission.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

A proteomic perspective on the changes in milk proteins due to high somatic cell count.

Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC.

Identification of unknown microcontaminants in Dutch river water by liquid chromatography-high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

In the past decade during automated surface water monitoring in the river Meuse at border station Eijsden in The Netherlands, a set of unknown compounds were repeatedly detected by online liquid chromatography-diode-array detection in a relatively high signal intensity. Because of the unknown nature of the compounds, the consequently unknown fate of this mixture in water treatment processes, the location being close to the water inlet of a drinking water supply company and their possible adverse public health effects, it was deemed necessary to elucidate the identity of the compounds. No data are available for the occurrence of these unknowns at downstream locations. After concentration and fractionation of a sample by preparative Liquid Chromatography, identification experiments were performed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HR-MS) combined with High Resolution Nuclear Magnetic Resonance Spectroscopy (HR-NMR). Accurate mass determination of the unknown parent compound and its fragments obtained in MS/MS provided relevant information on the elemental composition of the unknown compounds. With the use of NMR techniques and the information about the elemental composition, the identity of the compounds in the different sample fractions was determined. Beside some regularly detected compounds in surface water, like caffeine and bisphenol-S, five dihydroxydiphenylmethane isomers were identified. The major unknown compound was identified as 4,4'-dihydroxy-3,5,3',5'-tetra(hydroxymethyl)diphenylmethane. This compound was confirmed by analysis of the pure reference compound. This is one of the first studies that employs the combination of high resolution MS with NMR for identification of truly unknown compounds in surface waters at the µg/L level. Five of the seven identified compounds are unexpected and not contained in the CAS database, while they can be presumed to be products generated during the production of resins.

Quercetin decreases high-fat diet induced body weight gain and accumulation of hepatic and circulating lipids in mice.

Dietary flavonoids may protect against cardiovascular diseases (CVD). Increased circulating lipid levels and hepatic lipid accumulation are known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the flavonoid quercetin on hepatic lipid metabolism in mice with high-fat diet induced body weight gain and hepatic lipid accumulation. Adult male mice received a 40 energy% high-fat diet without or with supplementation of 0.33 % (w/w) quercetin for 12 weeks. Body weight gain was 29 % lower in quercetin fed mice (p < 0.01), while the energy intake was not significantly different. Quercetin supplementation lowered hepatic lipid accumulation to 29 % of the amount present in the control mice (p < 0.01). (1)H nuclear magnetic resonance serum lipid profiling revealed that the supplementation significantly lowered serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3), were downregulated. Quercetin decreased high-fat diet induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are possibly under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content of the diet.

High rates of intestinal colonisation with fluoroquinolone-resistant ESBL-harbouring Enterobacteriaceae in hospitalised patients with antibiotic-associated diarrhoea.

The purposes of this study were to investigate the intestinal carriage of extended-spectrum ß-lactamase-harbouring Enterobacteriaceae (ESBL-EN) and associated fluoroquinolone resistance (FQ-R) in 120 hospitalised patients with antibiotic-associated diarrhoea, and to investigate a correlation between Clostridium difficile (C. difficile) infection and intestinal colonisation with ESBL-EN in these patients. Stool samples were screened for C. difficile infection by toxin A/B enzyme-linked immunosorbent assay (ELISA) and for the presence of enterobacterial isolates producing ß-lactamases by plating on ß-lactamase screening (BLSE) agar. Recovered isolates were confirmed pheno- and genotypically for the presence of ESBL genes (bla CTX-M, bla TEM, bla SHV) by the double-disc synergy test and polymerase chain reaction (PCR) sequencing, and tested for the presence of topoisomerase mutations (gyrA, parC) and plasmid-mediated quinolone resistance (PMQR) determinants [qnrA, qnrB, qnrS, qepA, aac(6')-Ib-cr] by PCR sequencing. ESBL-EN were detected in 44/120 (37 %) stool samples. C. difficile-infected patients showed a significantly higher frequency of intestinal colonisation with ESBL-EN compared to C. difficile non-infected patients (62 % vs. 31 %, p = 0.008). Of the 73 ESBL-EN recovered, 46 (63 %) showed high-level FQ-R [ciprofloxacin minimum inhibitory concentration (MIC) ≥32 mg/L]. The largest group consisted of 21 bla CTX-M-15-harbouring Enterobacteriaceae (ciprofloxacin MIC ≥64 mg/L) with multiple topoisomerase mutations in gyrA and parC, in combination with co-carriage of aac(6')-Ib-cr. Most of them were Escherichia coli isolates belonging to sequence types ST131 and ST410. We found remarkably high rates of intestinal colonisation with high-level FQ-R ESBL-EN in hospitalised patients with antibiotic-associated diarrhoea, especially among C. difficile-infected patients. These data underscore the need for stringent infection control to contain this potentially infectious and multidrug-resistant reservoir.

Correlation between activation of PPARγ and resistin downregulation in a mouse adipocyte cell line by a series of thiazolidinediones.

The present study shows significant correlations between the EC50 for PPARγ activation in a reporter gene cell line and resistin downregulation in mouse adipocytes, and between the IC50 for resistin downregulation and the already published minimum effective dose for antihyperglycemic activity in a mouse model. These correlations indicate that PPARγ mediated downregulation of resistin might promote insulin sensitivity and that downregulation of resistin in mouse adipocytes provides an adequate and possibly more direct bioassay for screening of newly developed antihyperglycemic compounds. Because of the higher throughput of the PPARγ the resistin downregulation assays seems most suitable to be used as a second tier in a tiered screening strategy.

Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time.

Physiologically based biokinetic (PBBK) model for safrole bioactivation and detoxification in rats.

A physiologically based biokinetic (PBBK) model for alkenylbenzene safrole in rats was developed using in vitro metabolic parameters determined using relevant tissue fractions. The performance of the model was evaluated by comparison of the predicted levels of 1,2-dihydroxy-4-allylbenzene and 1'-hydroxysafrole glucuronide to levels of these metabolites reported in the literature to be excreted in the urine of rats exposed to safrole and by comparison of the predicted amount of total urinary safrole metabolites to the reported levels of safrole metabolites in the urine of safrole exposed rats. These comparisons revealed that the predictions adequately match observed experimental values. Next, the model was used to predict the relative extent of bioactivation and detoxification of safrole at different oral doses. At low as well as high doses, P450 mediated oxidation of safrole mainly occurs in the liver in which 1,2-dihydroxy-4-allylbenzene was predicted to be the major P450 metabolite of safrole. A dose dependent shift in P450 mediated oxidation leading to a relative increase in bioactivation at high doses was not observed. Comparison of the results obtained for safrole with the results previously obtained with PBBK models for the related alkenylbenzenes estragole and methyleugenol revealed that the overall differences in bioactivation of the three alkenylbenzenes to their ultimate carcinogenic 1'-sulfooxy metabolites are limited. This is in line with the generally less than 4-fold difference in their level of DNA binding in in vitro and in vivo studies and their almost similar BMDL(10) values (lower confidence limit of the benchmark dose that gives 10% increase in tumor incidence over background level) obtained in in vivo carcinogenicity studies. It is concluded that in spite of differences in the rates of specific metabolic conversions, overall the levels of bioactivation of the three alkenylbenzenes are comparable which is in line with their comparable carcinogenic potential.

Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism.

Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17ß-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.

Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect.

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1'-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1'-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1'-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 microM did not inhibit 1'-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1'-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1'-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio.

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.

A positive test of East Antarctica-Laurentia juxtaposition within the Rodinia supercontinent.

The positions of Laurentia and other landmasses in the Precambrian supercontinent of Rodinia are controversial. Although geological and isotopic data support an East Antarctic fit with western Laurentia, alternative reconstructions favor the juxtaposition of Australia, Siberia, or South China. New geologic, age, and isotopic data provide a positive test of the juxtaposition with East Antarctica: Neodymium isotopes of Neoproterozoic rift-margin strata are similar; hafnium isotopes of approximately 1.4-billion-year-old Antarctic-margin detrital zircons match those in Laurentian granites of similar age; and a glacial clast of A-type granite has a uraniun-lead zircon age of approximately 1440 million years, an epsilon-hafnium initial value of +7, and an epsilon-neodymium initial value of +4. These tracers indicate the presence of granites in East Antarctica having the same age, geochemical properties, and isotopic signatures as the distinctive granites in Laurentia.

[Fluorene degradation by bacteria of the genus Rhodococcus]. / Prevrashchenie fluorena bakteriiami roda Rhodococcus.

Of the four investigated Rhodococcus strains (R. rhodochrous 172, R. opacus 4a and 557, and R. rhodnii 135), the first three strains were found to be able to completely transform fluorene when it was present in the medium as the sole source of carbon at a concentration of 12-25 mg/l. At a fluorene concentration of 50-100 mg/l in the medium, the rhodococci transformed 50% of the substrate in 14 days. The addition of casamino acids and sucrose (1-5 g/l) stimulated fluorene transformation, so that R. rhodochrous 172 could completely transform it in 2-5 days. Nine intermediates of fluorene transformation were isolated, purified, and structurally characterized. It was found that R. rhodnii 135 and R. opacus strains 4a and 557 hydroxylated fluorene with the formation of 2-hydroxyfluorene and 2,7-dihydroxyfluorene. R. rhodochrous 172 transformed fluorene via two independent pathways to a greater degree than did the other rhodococci studied.

The molecular basis of co-evolution between Cladosporium fulvum and tomato.

Cladosporiumfulvum is a semi-biotrophic pathogen, which causes leaf mold of tomato (Lycopersicon spp.). In our laboratory this pathosystem serves as a model to study gene-for-gene interactions between plants and pathogenic fungi (Joosten & De Wit 1999). Many avirulence (Avr) genes and matching resistance (CQ) genes have been cloned and we are now beginning to understand how their products can induce an array of plant defense responses, including the classic hypersensitive response (HR). Here, we will discuss the latest results of our molecular studies on this interaction. These include the isolation of: (i) two new Avr genes, Avr2 and Avr4E, (ii) the determination of the specificity determinants within the Cf-4 and Cf-9 genes by artificial domain swaps and introduction of point mutations, (iii) the analysis of polymorphism occurring in AVR9-responsive Cf genes occurring in natural populations of L. pimpinellifolium, and finally (iv) the description of an efficient method to identify early HR-related genes.

Substrate interactions during the biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) hydrocarbons by the fungus Cladophialophora sp. strain T1.

The soil fungus Cladophialophora sp. strain T1 (= ATCC MYA-2335) was capable of growth on a model water-soluble fraction of gasoline that contained all six BTEX components (benzene, toluene, ethylbenzene, and the xylene isomers). Benzene was not metabolized, but the alkylated benzenes (toluene, ethylbenzene, and xylenes) were degraded by a combination of assimilation and cometabolism. Toluene and ethylbenzene were used as sources of carbon and energy, whereas the xylenes were cometabolized to different extents. o-Xylene and m-xylene were converted to phthalates as end metabolites; p-xylene was not degraded in complex BTEX mixtures but, in combination with toluene, appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that toluene, ethylbenzene, and xylene were degraded at the side chain by the same monooxygenase enzyme. Our findings suggest that soil fungi could contribute significantly to bioremediation of BTEX pollution.