Oliver J. Schmitz

Production of different phenotypes from the same genotype in the same environment by developmental variation

SUMMARY The phenotype of an organism is determined by the genes, the environment and stochastic developmental events. Although recognized as a basic biological principle influencing life history, susceptibility to diseases, and probably evolution, developmental variation (DV) has been only poorly investigated due to the lack of a suitable model organism. This obstacle could be overcome by using the recently detected, robust and highly fecund parthenogenetic marbled crayfish as an experimental animal. Batch-mates of this clonal crayfish, which were shown to be isogenic by analysis of nuclear microsatellite loci, exhibited surprisingly broad ranges of variation in coloration, growth, life-span, reproduction, behaviour and number of sense organs, even when reared under identical conditions. Maximal variation was observed for the marmorated coloration, the pattern of which was unique in each of the several hundred individuals examined. Variation among identically raised batch-mates was also found with respect to fluctuating asymmetry, a traditional indicator of the epigenetic part of the phenotype, and global DNA methylation, an overall molecular marker of an animals epigenetic state. Developmental variation was produced in all life stages, probably by reaction–diffusion-like patterning mechanisms in early development and non-linear, self-reinforcing circuitries involving behaviour and metabolism in later stages. Our data indicate that, despite being raised in the same environment, individual genotypes can map to numerous phenotypes via DV, thus generating variability among clone-mates and individuality in a parthenogenetic species. Our results further show that DV, an apparently ubiquitous phenomenon in animals and plants, can introduce components of randomness into life histories, modifying individual fitness and population dynamics. Possible perspectives of DV for evolutionary biology are discussed.

Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice.

Many studies using mammalian cellular and subcellular systems have demonstrated that polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic versus extra-hepatic metabolism of BaP and its pharmacokinetics, we used the hepatic cytochrome P450 reductase null (HRN) mouse model, in which cytochrome P450 oxidoreductase, the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated intraperitoneally (i.p.) with 125 mg/kg body wt BaP daily for up to 5 days. Clearance of BaP from blood was analysed by high-performance liquid chromatography with fluorescence detection. DNA adduct levels were measured by (32)P-post-labelling analysis with structural confirmation of the formation of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene by liquid chromatography-tandem mass spectrometry analysis. Hepatic microsomes isolated from BaP-treated and untreated mice were also incubated with BaP and DNA in vitro. BaP-DNA adduct formation was up to 7-fold lower with the microsomes from HRN mice than with that from WT mice. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A. Pharmacokinetic analysis of BaP in blood revealed no significant differences between HRN and WT mice. BaP-DNA adduct levels were higher in the livers (up to 13-fold) and elevated in several extra-hepatic tissues of HRN mice (by 1.7- to 2.6-fold) relative to WT mice. These data reveal an apparent paradox, whereby hepatic CYP enzymes appear to be more important for detoxification of BaP in vivo, despite being involved in its metabolic activation in vitro.

Efficient Synthesis of Carbazolyl- and Thienyl-Substituted β-Diketonates and Properties of Their Red- and Green-Light-Emitting Ir(III) Complexes

The efficient synthesis of novel beta-diketonates equipped with functional carbazolyl moieties and their subsequent transformations in 5-hexyl-thienyl substituted carbazole derivatives is presented by utilizing an effective Stille cross-coupling reaction. The introduced beta-diketonates served as ancillary ligands for novel heteroleptic red- and green-emitting Ir(III) complexes, when combined with 2-(naphthalen-1-yl)pyridine and 2-phenylpyridine as cyclometalating ligands. These novel Ir(III) complexes revealed color-tunability and a very good thermal stability until at least 207 degrees C. In polystyrene blends, the heteroleptic Ir(III) complexes revealed remarkable quantum yields up to 36% and suitably short phosphorescence lifetimes ranging from 1 to 4 micros. In the case of the orange-red Ir(III) emitter, equipped with 2-(naphthalen-1-yl)pyridine cyclometallating ligands, a luminous efficiency as high as 7.7 cd/A at 7.4 V was achieved. All fabricated diodes exhibited in addition favorable color stability.

Development of a multipurpose ion source for LC-MS and GC-API MS.

Over the past decade, multimode ion sources operating at atmospheric pressure (i.e., more than one ionization method is operative in the ion source enclosure) have received considerable interest. Simultaneous operation of different ionization methods targeting different compound classes within one analysis run has several advantages, including enhanced sample throughput and thus significant laboratory cost reductions. Potential drawbacks are enhanced ion suppression and other undesirable effects of the simultaneous operation of ionization methods. In this contribution we present an alternative approach—the development and characterization of a widely applicable, multipurpose ion source operating at atmospheric pressure. The optimized source geometry allows rapid changing from LC-API methods (ESI, APCI, APLI) to GC-API methods (APCI, APLI, DA-APLI) along with the appropriate coupling of chromatographic equipment required. In addition, true multimode operation of the source is demonstrated for LC-ESI/APLI and LC-APCI/APLI.

Tissue-Specific Effects of In Vitro Fertilization Procedures on Genomic Cytosine Methylation Levels in Overgrown and Normal Sized Bovine Fetuses

Abstract Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo–fertilized controls (n = 14) indicated an apparently normal phenotype for IVF1 fetuses (n = 5), but IVF2 fetuses (n = 10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (−16.1% and −9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

Analysis of catechins and caffeine in tea extracts by micellar electrokinetic chromatography.

Cancer chemotherapy is a new and important medical science and much interest has been focused on catechins, not only for their antioxidant activity, but also because of their known antimutagenic and antitumorigenic properties. Green tea and black tea, which are among the most popular beverages consumed worldwide, contain many different catechins. We developed an analytical method capable of separating six different catechins and caffeine in tea by micellar electrokinetic chromatography in only 20min without extensive sample preparation. Furthermore, we compared the amount of catechins and caffeine in several teas and different preparation modes.

Decrease in concentration of free catechins in tea over time determined by micellar electrokinetic chromatography

Cancer chemoprevention is a new and important medical science and much interest has been focused on catechins, not only for their antioxidant activity, but also because of their known antimutagenic and antitumorigenic properties. Green tea and black tea, which are among the most popular beverages consumed worldwide, contain many different catechins. Due to the instability of catechins in solutions with neutral or basic pH values the concentrations of catechins in tea decrease in time. In this presentation we used micellar electrokinetic chromatography to determine the real concentration of catechins between 0 and 60 min after the tea was brewed.

Combining chip-ESI with APLI (cESILI) as a multimode source for analysis of complex mixtures with ultrahigh-resolution mass spectrometry

Recently we have established atmospheric-pressure laser ionisation (APLI) as a method for coupling time-of-flight mass spectrometric detectors (TOF MS) with chromatographic systems (HPLC and GC) to allow two-photon ionisation of non-polar aromatic compounds. Here we demonstrate that APLI can be combined with chip-electrospray ionisation (cESI) coupled to Fourier-transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) for ultrahigh-resolution analysis of complex samples. With the laser turned off, the analytes are ionised only by ESI, whereas when the laser is switched on non-polar aromatic substances also are ionised. In combination with the extremely high mass resolution of an FT-ICR MS, simultaneous qualitative analysis of polar and non-polar analytes is possible in both positive and negative modes, as is exemplified with a crude oil sample. Nevertheless, ion suppression was observed (up to ca. 70% for D10-pyrene) and thus sample preparation with chromatographic or electrophoretic pre-separation is necessary for quantitative analysis of targets. In addition, for the first time, the dopant-assisted APLI method in combination with cESI (DA-cESILI) was used for determination of 1-nitrocoronene.

High-Performance Liquid Chromatography–Mass Spectrometry Profiling of Phenolic Compounds for Evaluation of Olive Oil Bitterness and Pungency

Bitterness and pungency are important parameters for olive oil quality. Therefore, two instrumental methods for evaluation of these taste attributes were developed. The first one is based on the photometric measurement of total phenolic compounds content, whereas the second one is based on the semiquantitative evaluation of hydrophilic compounds by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Evaluation of total phenolic compounds content was performed by a modified method for the determination of the K(225) value using a more specific detection based on the pH value dependency of absorbance coefficients of phenols at λ = 274 nm. The latter method was not suitable for correct prediction, because no significant correlation between bitterness/pungency and total phenolic compounds content could be found. For the second method, areas of 25 peaks detected in 54 olive oil samples by a HPLC-MS profiling method were correlated with the bitterness and pungency by partial least-squares regression. Six compounds (oleuropein aglycon, ligstroside aglycon, decarboxymethyl oleuropein aglycon, decarboxymethyl ligstroside aglycon, elenolic acid, and elenolic acid methyl ester) show high correlations to bitterness and pungency. The computed model using these six compounds was able to predict bitterness and pungency of olive oil in the error margin of the sensory evaluation (±0.5) for most of the samples.

Comprehensive two-dimensional liquid chromatography tandem diode array detector (DAD) and accurate mass QTOF-MS for the analysis of flavonoids and iridoid glycosides in Hedyotis diffusa

The analysis of chemical constituents in Chinese herbal medicines (CHMs) is a challenge because of numerous compounds with various polarities and functional groups. Liquid chromatography coupled with quadrupole time-of-flight (QTOF) mass spectrometry (LC/MS) is of particular interest in the analysis of herbal components. One of the main attributes of QTOF that makes it an attractive analytical technique is its accurate mass measurement for both precursor and product ions. For the separation of CHMs, comprehensive two-dimensional chromatography (LCxLC) provides much higher resolving power than traditional one-dimensional separation. Therefore, a LCxLC-QTOF-MS system was developed and applied to the analysis of flavonoids and iridoid glycosides in aqueous extracts of Hedyotis diffusa (Rubiaceae). Shift gradient was applied in the two-dimensional separation in the LCxLC system to increase the orthogonality and effective peak distribution area of the analysis. Tentative identification of compounds was done by accurate mass interpretation and validation by UV spectrum. A clear classification of flavonol glycosides (FGs), acylated FGs, and iridoid glycosides (IGs) was shown in different regions of the LCxLC contour plot. In total, five FGs, four acylated FGs, and three IGs were tentatively identified. In addition, several novel flavonoids were found, which demonstrates that LCxLC-QTOF-MS detection also has great potential in herbal medicine analysis.