Downregulation of CENPF Remodels Prostate Cancer Cells and Alters Cellular Metabolism

Metabolic alterations in prostate cancer (PC) are associated with progression and aggressiveness. However, the underlying mechanisms behind PC metabolic functions are unknown. The authors’ group recently reported on the important role of centromere protein F (CENPF), a protein associated with the centromere–kinetochore complex and chromosomal segregation during mitosis, in PC MRI visibility. This study focuses on discerning the role of CENPF in metabolic perturbation in human PC3 cells. A series of bioinformatics analyses shows that CENPF is one gene that is strongly associated with aggressive PC and that its expression is positively correlated with metastasis. By identifying and reconstructing the CENPF network, additional associations with lipid regulation are found. Further untargeted metabolomics analysis using gas chromatography‐time‐of‐flight‐mass spectrometry reveals that silencing of CENPF alters the global metabolic profiles of PC cells and inhibits cell proliferation, which suggests that CENPF may be a critical regulator of PC metabolism. These findings provide useful scientific insights that can be applied in future studies investigating potential targets for PC treatment.     

Synthesis of novel D-ring fused 7'-aryl-androstano[17,16-d][1,2,4] triazolo[1,5-a]pyrimidines

The preparation of novel steroidal heterocycles containing the 7-aryl-substituted 1,2,4-triazolo[1,5-a]pyrimidine moiety fused to the 16,17-positions of the steroid nucleus is described. The Aldol reaction of 4-aza-androst-3,17-dione (1a) and dehydroepiandrosterone (DHEA, 1b) with aromatic aldehydes was catalyzed by KF/Al(2)O(3) to give the corresponding 3-oxo-4-aza-5α- and 3β-hydroxy-5-en-16-arylidene-17-ketosteroids (2a-r). Subsequently, the intermediates 2a-r reacted with dinucleophilic 3-amino-1,2,4-triazole in presence of t-BuOK to afford the title compounds (3a-r). All the synthesized heterosteroids are new and are currently being evaluated for their biological activities.     

Recurrent Tissue-Specific mtDNA Mutations Are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.     

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca²⁺‐sensitive dye Fura2‐AM was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca²⁺]_i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca²⁺‐activated Cl^? channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl^? secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of the ATP·Mg-dependent protein phosphatase activator (Fa ) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules

The ATP·Mg-dependent protein phosphatase activating factor (Fa) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factorFa has further been identified as a cAMP and Ca²⁺-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factorFa could phosphorylate synapsin I with a lowK _m value of about 2 µM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factorFa specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factorFa could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factorFa as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.     

Post‐activation treatment with demecolcine improves development of somatic cell nuclear transfer embryos in pigs by modifying the remodeling of donor nuclei

The objective of this study was to examine the effect of cytochalasin B (CB) and/or demecolcine (Dc) on the remodeling of donor nuclei, nuclear ploidy, and development of somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) pig embryos. SCNT and PA oocytes were either untreated (control), or treated with CB, Dc, or both CB and Dc after electric activation, and then cultured or transferred to surrogates. In SCNT, blastocyst formation was higher after treatment with CB and/or Dc (26–28%) than in the controls (16%). The number of oocytes that formed a single pronucleus (PN) was higher after treatment with Dc (86%) and CB + Dc (86%) than under control conditions (44%) or after treatment with CB (63%). In PA, blastocyst formation was higher after CB treatment (47%) than under control conditions (28%), while the formation of a single PN was higher after treatment with Dc (88%) and CB + Dc (84%) compared to controls (34%). The rate of formation of diploid embryos was higher after treatment with Dc and CB + Dc than under control conditions. Dc treatment resulted in a farrowing rate of 50% with 1.1% production efficiency, while controls showed a farrowing rate of 37.5% and a production efficiency of 0.7%. The results of our study demonstrate that post‐activation treatment with Dc improves preimplantation development and supports normal in vivo development of SCNT pig embryos, probably because Dc induces formation of a single PN and this leads to normal nuclear ploidy. Mol. Reprod. Dev. 76: 611–619, 2009. © 2008 Wiley‐Liss, Inc.     

Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor

The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes, ranging from axon guidance and nerve regeneration to cancer malignancy. Thus antagonists that inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic applications. In the present study we characterize the binding features of three antagonistic peptides (KYL, APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration calorimetry analysis demonstrated that all three peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4. Chemical-shift changes observed by NMR spectroscopy upon binding of the KYL peptide involve many EphA4 residues, consistent with extensive interactions and possibly receptor conformational changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM identify the residues critical for EphA4, binding. The peptides exhibit a long half-life in cell culture medium which, with their substantial binding affinity and selectivity for EphA4, makes them excellent research tools to modulate EphA4 function.     

Formation mechanism of cross‐linking Maillard compounds in peptide–xylose systems

The formation mechanism of Maillard peptides was explored in Maillard reaction through diglycine/glutathione(GSH)/(Cys‐Glu‐Lys‐His‐Ile‐Met)–xlyose systems by heating at 120 °C for 30–120 min. Maximum fluorescence intensity of Maillard reaction products (MRPs) with an emission wavelength of 420~430 nm in all systems was observed, and the intensity values were proportional to the heating time. Taken diglycine/GSH–[¹³C₅]xylose systems as a control, it was proposed that the compounds with high m/z values of 379 and 616 have the high molecular weight (HMW) products formed by cross‐linking of peptides and sugar. In (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the m/z value of HMW MRPs was not observed, which might be due to the weak signals of these products. According to the results of gel permeation chromatography, HMW MRPs were formed by Maillard reaction, especially in (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the percentage of Maillard peptides reached 52.90%. It was concluded that Maillard peptides can be prepared through the cross‐linking of sugar and small peptides with a certain MW range. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.