Relative expression of functional genes of CYP3A subfamily members in human brain samples.

<p>(a) The levels of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 mRNA were quantified by qRT-PCR in the 6 human brain subjects. The expression of CYP3A5 was significantly lower then that of CYP3A4 and CYP3A43 and was detected only in four out of six samples. CYP3A7 was not detected in any of the subjects. (b) The average of the six samples analyzed.</p

Metabolism of alprazolam to α-OHALP and 4-OHALP by human brain, in vitro.

<p>The expression levels of CYP3A4 and CYP3A43 were compared with the rates of formation of α-OHALP and 4-OHALP in samples of cortex obtained from 6 human brain subjects. Subjects represented in (a, b, c) expressed high levels of CYP3A43 and relatively higher amounts of α-OHALP was formed compared to 4-OHALP (g, h, i). Subjects (d, e) expressed high levels of CYP3A4 and formed relatively higher amounts of 4-OHALP (j, k). Subject (f) showed similar levels of expression CYP3A4 and CYP3A43 which was reflected in the amount of hydroxylated metabolite formed. Pearson correlation analysis showed high degree of correlation between the expression levels of CYP3A43 and amounts of α-OHALP formed (m; r = 0.74).</p

Constitutive expression of CYP3A43 mRNA in human brain cortex.

<p>(a) Total RNA was extracted from human brain cortex of 2 subjects and the 3′-UTR of CYP3A43 was amplified by RT-PCR (lanes 1 and 2). Lane N denoted reaction performed without template DNA. The size of the PCR product was 85bp. (b) Total RNA from human brain cortex was electrophoresed, transferred to positively charged nylon membrane and hybridized with antisense riboprobe prepared using the partial cDNA to CYP3A43. The mobility of 18S and 28S ribosomal RNA on the agarose gel is indicated.</p

Amplification of exonic regions of the ORF of CYP3A43 and CYP3A4 mRNA by RT-PCR.

<p>(a) The schematic representation of 13 exons of CYP3A43 along with their splice variants detected by RT-PCR is depicted. (b) A 432bp amplicon was amplified spanning the exons 1–5 (lanes 1 and 2). (c) Two amplicons (594bp and 450bp) were generated when exons 6–10 were amplified (lanes 1 and 2). The 594bp product was the expected amplicon while the other amplicon (450bp) was a mixture of two PCR products of which one had a deletion of exon 7 and other had a deletion of exon 8. (d) Amplification of the region spanning exons 11–13 gave two amplicons of sizes 486bp and 350bp. The 486bp product was the expected size while 350bp product represented a splice variant that had the deletion of exon 12. (e) Amplification of exons 6–13 generated an amplicon (950bp) representing a splice variant having deletion of both exon 7 and 8 and partial inclusion of 121bp intron 7.</p

Localization of <i>CYP3A43</i> mRNA in human brain by fluorescence in situ hybridization.

<p>(a) Intense fluorescence was seen in neuronal cell layers of frontal cortex. Differential localization was seen delineating the laminar architecture of the cortex. Bar = 100 µm (b) Higher magnification of cortical neurons (arrow). Bar = 25 µm (c) Control section hybridized with sense probe did not show any fluorescence. Bar = 100 µm (d) Reticular neurons in the midbrain expressed CYP3A43 mRNA. Bar = 100 µm (e) Fluorescent labelling of the granule cell layer (GL) in human cerebellum. Sparse staining was observed in the molecular layer (ML). Bar = 100 µm (f) Higher magnification of neurons of the granule cell layer in cerebellum. Bar = 25 µm (g) Control section hybridised with sense probe. Bar = 100 µm (h) CYP3A43 expression was seen in pyramidal neurons of CA1 in the hippocampus. Bar = 50 µm (i) Higher magnification of CA1 neurons. Bar = 25 µm (j) Robust staining of the CA2 pyramidal cell layer (arrow) of the hippocampus. Bar = 50 µm (k) Higher magnification of pyramidal neurons in the CA2 subfield. Bar = 25 µm (l) Intense fluorescence was seen in the CA3 pyramidal neurons (arrow head) of the human brain. Bar = 50 µm (m) Higher magnification of CA3 neurons (arrow head). Bar = 25 µm (n) Intense fluorescence was observed in the dentate gyrus. Staining of the interneurons of the hilus (arrow head) was also observed. Bar = 100 µm (o) Higher magnification of the granule cells in the dentate gyrus. Bar = 25 µm (p) Control section of hippocampus hybridized with sense probe did not show any fluorescence. Bar = 100 µm</p

Quantitative assessment of the expression of CYP3A4 and CYP3A43 in human brain cortex.

<p>(a) RT-PCR amplification of the 85bp of 3′-UTR of CYP3A43 and 78bp of 3′-UTR of CYP3A4 using human brain and liver RNA. (b) qRT-PCR analysis using human liver and brain RNA (n = 3) showed the relative higher expression of CYP3A43 in brain compared to liver from the same individuals. Quantitation of the relative expression of CYP3A4 and CYP3A43 in (c) different human brain tissues (n = 1) and (d) from different regions of the brain from same individual (n = 3).</p

Cloning and expression CYP3A43 and CYP3A4 and metabolism of alprazolam, in vitro by recombinant enzymes.

<p>The complete open reading frame of CYP3A43 and CYP3A4 were amplified by RT-PCR using total RNA from four autopsy human brain samples. The amplicons of 1586bp and 1512bp were generated for CYP3A4 and CYP3A43, respectively (a, and b, from lanes 1–4). M represents 100bp ladder and Ne, the control reaction performed without template DNA. The amplicons were cloned into pcDNA 3.1 for expression in COS-1 cells. (c) Recombinant CYP3A43 metabolized alprazolam to α-OHALP and 4-OHALP in similar amounts (26 pmoles/min/mg protein), while recombinant CYP3A4 metabolized alprazolam only to 4-OHALP. The values are mean ISD (n = 3 independent experiments). (d) Immunoblot of recombinant CYP3A43 and CYP3A4 normalized using β-tubulin.</p

Fibroblast characterization: FSP-1 protein expression by immunofluorescence staining and cell proliferation assay in dura and scalp. A.

<p>The morphology of postmortem fibroblast cells generated from (a) dura and (b) scalp. Cultured cells from both sources macroscopically looked similar to what is seen in living skin fibroblast cells, with more enriched cytoplasm and spindle-shaped nuclei under phase-contrast microscopy. Cells from (c) dura and (d) scalp express cytoplasmic Fibroblast Specific Protein-1 (FSP-1) (green). Original scale bars = 35 µm. <b>B.</b> Results from cell proliferation assay in 8 fibroblast cell lines (dura and scalp from 4 individuals) in five different densities. Cell viability was determined in 24 hrs and 48 hrs by WST-8 assay. Values are the mean of results from six wells. Bars ± SE. Scalp fibroblast cell lines grew 1.27-fold faster in the same period than dura fibroblast cells. <b>C.</b> Differences in cell proliferation between scalp and dura by one-way ANOVA; scalp cell growth was significantly more rapid than dura cell growth at 24 hr and 48 hr intervals [F (1, 46) = 12.94, p<0.008].</p

iPSCs generated from one dura fibroblast line express pluripotency markers and differentiate to neuronal fates.

<p>Undifferentiated iPSCs express pluripotency markers NANOG (A) and SOX2 (B). Upon neural differentiation, these cells express neuroectoderm marker SOX1 (C). Temporal gene expression analysis also shows a decrease in pluripotency marker OCT4 (POU5F1) and an increase in <i>PAX6</i> expression by quantitative RT-PCR (D). iPSC-derived neurons express βIII-tubulin and MAP2 (E–G).</p

Demographic and sample parameters of successful culture in scalp (n = 146).

<p>OR = odds ratio, AA = African-American, W = White, PMI = postmortem interval, BMI = body mass index, F = female, M = male, VA = Virginia, DC = District of Columbia; Tox = toxicology testing in blood or vitreous humor; * = p<0.05.</p