The protein tyrosine phosphatase Pez regulates TGFβ, epithelial–mesenchymal transition, and organ development

Epithelial–mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFβ signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFβ3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFβ and EMT

Single-molecule imaging of DNA gyrase activity in living Escherichia coli

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ~12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ~2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ~8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome

Common variants at ABCA7, MS4A6A/MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer's disease

We sought to identify new susceptibility loci for Alzheimer's disease through a staged association study (GERAD+) and by testing suggestive loci reported by the Alzheimer's Disease Genetic Consortium (ADGC) in a companion paper. We undertook a combined analysis of four genome-wide association datasets (stage 1) and identified ten newly associated variants with P ≤ 1 × 10−5. We tested these variants for association in an independent sample (stage 2). Three SNPs at two loci replicated and showed evidence for association in a further sample (stage 3). Meta-analyses of all data provided compelling evidence that ABCA7 (rs3764650, meta P = 4.5 × 10−17; including ADGC data, meta P = 5.0 × 10−21) and the MS4A gene cluster (rs610932, meta P = 1.8 × 10−14; including ADGC data, meta P = 1.2 × 10−16) are new Alzheimer's disease susceptibility loci. We also found independent evidence for association for three loci reported by the ADGC, which, when combined, showed genome-wide significance: CD2AP (GERAD+, P = 8.0 × 10−4; including ADGC data, meta P = 8.6 × 10−9), CD33 (GERAD+, P = 2.2 × 10−4; including ADGC data, meta P = 1.6 × 10−9) and EPHA1 (GERAD+, P = 3.4 × 10−4; including ADGC data, meta P = 6.0 × 10−10)

Structural determinants of the partial agonist-inverse agonist properties of 6′-azidohex-2′-yne-Δ(8)-tetrahydrocannabinol at cannabinoid receptors

1. We have extended previous investigations of four analogues of Δ(8)-tetrahydrocannabinol (Δ(8)-THC): 6′-azidohex-2′-yne-Δ(8)-THC (O-1184), 6′-azidohex-cis-2′-ene-Δ(8)-THC (O-1238) and octyl-2′-yne-Δ(8)-THC (O-584) and its 1-deoxy-analogue (O-1315). 2. O-1184, O-1238 and O-584 displaced [(3)H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB(1) or CB(2) cannabinoid receptors, with pK(i) values of 8.28 to 8.45 (CB(1)) and 8.03 to 8.13 (CB(2)). The pK(i) values of O-1315 were significantly less, 7.63 (CB(1)) and 7.01 (CB(2)). 3. All the analogues inhibited forskolin-stimulated cyclic AMP production by CB(1)-transfected CHO cells (pEC(50)=9.16 to 9.72). Only O-1238 behaved as a full agonist in this cell line. 4. In mouse vasa deferentia, O-1238 inhibited electrically-evoked contractions (pEC(50)=10.18 and E(max)=70.5%). Corresponding values for O-1184 were 9.08 and 21.1% respectively. At 1 nM, O-1184 produced surmountable antagonism of the cannabinoid receptor agonist, CP55940. However, at 0.1 nM, O-1184 did not attenuate CP55940-induced inhibition of cyclic AMP production by CB(1)-transfected CHO cells. 5. In CB(2)-transfected CHO cells, cyclic AMP production was inhibited by CP55940 (pEC(50)=8.59), enhanced by O-1184 and O-584 (pEC(50)=8.20 and 6.86 respectively) and not significantly affected by O-1238 or O-1315. 6. At 100 nM, O-1184 and O-1238 produced surmountable antagonism of CP55940 in CB(2) cells, decreasing the pEC(50) of CP55940 from 8.61 to 7.42 (O-1184) or from 8.54 to 7.44 (O-1238). 7. These data support the hypothesis that increasing the degree of unsaturation of the aliphatic side-chain of Δ(8)-THC analogues has little effect on CB(1) or CB(2) receptor affinity but can reduce CB(1) receptor efficacy and reverse the direction of responses elicited at CB(2) receptors