Annotation and classification of serum metabolites with high relevance in the classification of temperament types.

<p>Annotation and classification of serum metabolites with high relevance in the classification of temperament types.</p

Principal component score plot for prefrontal cortex and serum metabolites.

<p>Principal component score plot of the first two principal components for (A) the complete prefrontal cortex data and (B) the complete serum data. Symbols indicate the different temperament types, rhombs the fearful/neophobic-alert, circles the interested-stressed, triangles the outgoing/neophilic-alert, squares the subdued/uninterested-calm and crosses the indistinct temperament type.</p

Annotation and classification of prefrontal cortex metabolites with high relevance in the classification of temperament types.

<p>Annotation and classification of prefrontal cortex metabolites with high relevance in the classification of temperament types.</p

Significant differences between temperament types in the abundance of selected excitatory and inhibitory neurotransmitters in the prefrontal cortex.

<p>Significant differences (<i>p</i> < 0.05) between temperament types are indicated by arrows.</p><p>Significant differences between temperament types in the abundance of selected excitatory and inhibitory neurotransmitters in the prefrontal cortex.</p

Metabolites with high relevance in the classification of temperament types.

<p>Metabolite names, measuring technique and their VIP-Scores, number of occurrences in the outer cross-validation runs and nominal <i>p</i>-values for the Kruskal-Wallis-Test are shown for serum (S) and prefrontal cortex (PC) metabolites with high relevance in the classification of temperament types.</p><p>Metabolites with high relevance in the classification of temperament types.</p

Hierarchical clustering of metabolites with high relevance.

<p>Two way hierarchical clustering of (A) prefrontal cortex and (B) serum metabolite features with a high relevance in the classification of temperament types. Symbols indicate the different temperament types, rhombs the fearful/neophobic-alert, circles the interested-stressed, triangles the outgoing/neophilic-alert, squares the subdued/uninterested-calm and crosses the indistinct temperament type.</p

Polarisation of IPEC-1 and IPEC-J2, confocal microscopy and transmission electron microscopy.

<p>(A) Ten days old IPEC-1 and IPEC-J2 cells were fixed and stained for ZO-1 (<i>Zonula occludens</i>, green) and beta-catenin (<i>Zonula adherens</i>, red). Both cells lines showed a polarised structure with ZO-1 immunoreactivity at the apical pole and the underlying beta-catenin expression. (B) Important genes of a polarised brush border were analysed using microarray and qPCR. Here, significant differences were found between the cell lines. VIL1 (villin-1), VIL2 (villin-2 = ezrin), TLR4 (toll like receptor 4), MUC4 (mucin 4) and ESPN (espin) were significant up-regulated in IPEC-J2 in comparison to IPEC-1 in microarray as well as in qPCR. Villin1 and villin2 (= ezrin) were followed up by Western blot analyses. No villin-1 was detected in IPEC-1 but a strong expression in IPEC-J2 over time. Villin-2 was expressed in both cell lines. (C) Cells were cultured for 10 or 31 days and analysed using transmission electronmicroscopy. Both cell lines showed well-developed tight junctions at day 10 and 31 (red arrows). Desmosomes (blue arrows) and interdigitation (yellow arrow) were also observed. No differences were detected concerning microvilli length between both cell lines and at both time points. In contrast, the number of microvilli differed between the cell lines (IPEC-J2>IPEC-1).</p

Anchorage independent growth.

<p>Both cell lines were seeded in “Soft agar” (S, 0.33%) on “Feeder agar” (F, 0.5%) with (w) or without (wo) the application of EGF and ITS. Caco-2 cells were used as positive control. No anchorage independent growth was detected in IPEC-1 and IPEC-J2. On the other hand, Caco-2 showed an anchorage independent growth which did not depend on the additives EGF and ITS (bar = 20 μm). The results represent at least three independent experiments (n = 3).</p

Analysis of the protein expression of tight junctions and cytoskeleton—IPEC-1.

<p>Different proteins of the tight junctions and cytoskeleton were analysed by Western blot and immunofluorescence: CK18, β-actin, ZO-1, occludin, claudin-3 and claudin-4. At day 2 of culture IPEC-1 showed in the Western blots a weak protein expression of all proteins studied. A strong ZO-1 and occludin immunoreactivity was found at the border of the cells. In IPEC-1 ZO-1 showed a low expression, whereas a large amount of occludin was present. The cytoskeleton proteins CK18 and actin were strongly expressed at the border of the IPEC-1 cells. Furthermore, no stress fibres were detected in the cells. Claudin-3 and claudin-4 were observed in the cells. A spot like character of claudin-3 distribution in the area where at least three cells were closely located. Claudin-4 showed an expression at the cell border but also within the cytoplasm. (blue = DAPI; bar = 20 μm)</p

Analyses of important genes of the metabolism and oxygen-consumption.

<p>Important genes of the metabolism were analysed in both cell lines cultured on membranes for 10 days using microarray analyses and qPCR (A). PDHB (pyruvate dehydrognase subunit B) and CYC1 (cyctochrome C) are significantly down-regulated in the microarray and qPCR. SDH (succinate dehydrogenase subunit B) and HIF1a (hypoxia inducible factor 1a) are both significantly up-regulated in the microarray but not in qPCR. Furthermore, oxygen-consumption of both cell lines cultured on dishes or membranes for 10 days was examined. Both cell lines showed a significant higher O₂-consumption on membranes (IPEC-1: 20.07 nmol/100 000 cells; IPEC-J2: 75.27 nmol/100 000 cells) in comparison to dishes (IPEC-1: 3.18 nmol/100 000 cells; IPEC-J2: 8.18 nmol/100 000 cells). At the same time, a significant higher oxygen-consumption was found in IPEC-J2 in comparison to IPEC1, which was independent of the culture support.</p