New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples

The application of a real-time quantitative PCR method (5' nuclease assay), based on the use of a probe labeled at its 5' end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 x 10(4) cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Hormonal responses to a resistance exercise performed under the influence of delayed onset muscle soreness

Hormonal responses to an unaccustomed knee-extension exercise (E1; 5 times 10 repetitions with 40% load of 1RM [1 repetition maximum] followed by 2 sets until exhaustion) were compared in 6 men with the corresponding responses to an identical exercise performed 2 days later under the influence of delayed onset muscle soreness (DOMS) (E2). Both exercises were performed with a variable-resistance machine causing exhaustion with significantly fewer repetitions than a normal constant-resistance knee-extension device does. The E1 induced DOMS as expected, but the 1RM, the total work done, and the repetition number and frequency were not different in the 2 exercises. In the 2 sets to failure, the mean repetition number varied between 17 and 25. The exercise-induced norepinephrine, epinephrine, testosterone, cortisol (COR), and growth hormone (GH) increases were similar in the 2 exercises, although the overall level of COR and GH, including the preexercise concentrations, tended to decline in the second exercise. The results may thus suggest that the hormonal response to resistance exercise is not significantly altered when performed soon after an unaccustomed exercise bout leading to DOMS.     

Application of centrifugal force to the extraction and separation of parasorboside and gerberin from Gerbera hybrida

A method for the separation of parasorboside and gerberin from the ornamental plant Gerbera hybrida (Asteraceae) has been developed. The two closely related glucosides were extracted using an Extrachrom instrument, a prototype multi-functional separation tool equipped with an extraction chamber. The rotation planar extraction procedure was compared with that of a medium pressure solid-liquid extraction system. The resulting extracts were pre-purified using rotation planar chromatography and the results compared with those obtained using medium pressure liquid chromatography with silica gel as the stationary phase and a mobile phase of methanol:ethyl acetate:tetrahydrofuran at selectivity point Ps = 111 with 1% formic acid as modifier. The title compounds were isolated from the purified extracts by TLC and their structures confirmed by 1H- and 13C-NMR spectroscopy.     

Heterologous expression of dihydroflavonol 4-reductases from various plants

Dihydroflavonol 4-reductases (DFR) catalyze the stereospecific reduction of dihydroflavonols to the respective flavan 3,4-diols (leucoanthocyanidins) and might also be involved in the reduction of flavanones to flavan-4-ols, which are important intermediates in the 3-deoxyflavonoid pathway. Several cDNA clones encoding DFR have been isolated from different plant species. Despite the important function of these enzymes in the flavonoid pathway, attempts at heterologous expression of cDNA clones in Escherichia coli have failed so far. Here, three well known heterologous expression systems for plant-derived genes were tested to obtain the functional protein of DFR from Gerbera hybrids. Successful synthesis of an active DFR enzyme was achieved in eukaryotic cells, using either baker's yeast (Saccharomyces cerevisiae) or tobacco protoplasts (Nicotiana tabacum), transformed with expression vectors containing the open reading frame of Gerbera DFR. These expression systems provide useful and powerful tools for rapid biochemical characterization, in particular the substrate specificity, of the increasing number of cloned DFR sequences. Furthermore, this tool allows the stereospecific synthesis of (14)C-labeled leucoanthocyanidins in high quality and quantity, which is a prerequisite for detailed biochemical investigation of the less understood enzymatic reactions located downstream of DFR in anthocyanin, catechin and proanthocyanidin biosynthesis.     

Altered Activation of Innate Immunity Associates with White Matter Volume and Diffusion in First-Episode Psychosis

First-episode psychosis (FEP) is associated with inflammatory and brain structural changes, but few studies have investigated whether systemic inflammation associates with brain structural changes in FEP. Thirty-seven FEP patients (median 27 days on antipsychotic medication), and 19 matched controls were recruited. Serum levels of 38 chemokines and cytokines, and cardiovascular risk markers were measured at baseline and 2 months later. We collected T1- and diffusion-weighted MRIs with a 3 T scanner from the patients at baseline. We analyzed the association of psychosis-related inflammatory markers with gray and white matter (WM) volume using voxel-based morphometry and WM diffusion using tract-based spatial statistics with whole-brain and region-of-interest (ROI) analyses. FEP patients had higher CCL22 and lower TGFα, CXCL1, CCL7, IFN-α2 and ApoA-I than controls. CCL22 decreased significantly between baseline and 2 months in patients but was still higher than in controls. The association between inflammatory markers and FEP remained significant after adjusting for age, sex, smoking and BMI. We did not observe a correlation of inflammatory markers with any symptoms or duration of antipsychotic treatment. Baseline CCL22 levels correlated negatively with WM volume and positively with mean diffusivity and radial diffusivity bilaterally in the frontal lobes in ROI analyses. Decreased serum level of ApoA-I was associated with smaller volume of the medial temporal WM. In whole-brain analyses, CCL22 correlated positively with mean diffusivity and radial diffusivity, and CXCL1 associated negatively with fractional anisotropy and positively with mean diffusivity and radial diffusivity in several brain regions. This is the first report to demonstrate an association between circulating chemokine levels and WM in FEP patients. Interestingly, CCL22 has been previously implicated in autoimmune diseases associated with WM pathology. The results suggest that an altered activation of innate immunity may contribute to WM damage in psychotic disorders.     

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.