Frame-shifted APOBEC3A encodes two alternative proapoptotic proteins that target the mitochondrial network

The human APOBEC3A (A3A) cytidine deaminase is a powerful DNA mutator enzyme recognized as a major source of somatic mutations in tumor cell genomes. However, there is a discrepancy between APOBEC3A mRNA levels after interferon stimulation in myeloid cells and A3A detection at the protein level. To understand this difference, we investigated the expression of two novel alternative “A3Alt” proteins encoded in the +1-shifted reading frame of the APOBEC3A gene. A3Alt-L and its shorter isoform A3Alt-S appear to be transmembrane proteins targeted to the mitochondrial compartment that induce membrane depolarization and apoptosis. Thus, the APOBEC3A gene represents a new example wherein a single gene encodes two proapoptotic proteins, A3A cytidine deaminases that target the genome and A3Alt proteins that target mitochondria.     

Bio-guided search of active indole alkaloids from Tabernaemontana catharinensis: Antitumour activity,toxicity in silico and molecular modelling studies

Active plant metabolites have been used as prototype drugs. In this context, Tabernaemontana catharinensis (Apocynaceae) has been highlighted because of the presence of active indole alkaloids. Thus, this study aims the bio-guided search of T. catharinensis cytotoxic alkaloids. The chemical composition was identified by high-resolution mass spectrometry, and fractionation was performed by open column and preparative thin-layer chromatography, from plant stems. The enriched fractions were tested in vitro in tumour cells A375 (melanoma cell line) and A549 (adenocarcinomic human alveolar basal epithelial cells), and non-tumour Vero cells (African green monkey kidney epithelial cells). The alkaloids identified as active were submitted to in silico toxicity prediction by ADME-Tox and OSIRIS programs and, also, to molecular docking, using topoisomerase I (PDB ID: 1SC7) by iGEMDOCK. As a result, six sub-fractions were obtained, which were identified as containing 16-epi-affinine, 12-methoxy-n-methyl-voachalotine, affinisine, voachalotine, coronaridine hydroxyindoline and ibogamine, respectively. The affinisine-containing sub-fraction showed selective toxicity against A375, with an IC₅₀ of 11.73 µg mL⁻¹, and no cytotoxicity against normal cells (Vero). From the in silico toxicity test results, all indole alkaloid compounds had a low toxicity risk. The molecular docking data provided structural models and binding affinities of the plant’s indole alkaloids and topoisomerase I. In summary, this bio-guided search revealed that the indole alkaloids from T. catharinensis display selective cytotoxicity in A375 tumour cells and toxicity in silico. Particularly, affinisine might be a chemotherapeutic for A375 melanoma cells.     

Distinct properties of cyclin-dependent kinase complexes containing cyclin A1 and cyclin A2

The distinct expression patterns of the two A-type cyclins during spermatogenesis and the absolute requirement for cyclin A1 in this biological process in vivo suggest that they may confer distinct biochemical properties to their CDK partners. We therefore compared human cyclin A1- and cyclin A2-containing CDK complexes in vitro by determining kinetic constants and by examining the complexes for their ability to phosphorylate pRb and p53. Differences in biochemical activity were observed in CDK2 but not CDK1 when complexed with cyclin A1 versus cyclin A2. Further, CDK1/cyclin A1 is a better kinase complex for phosphorylating potentially physiologically relevant substrates pRb and p53 than CDK2/cyclin A2. The activity of CDKs can therefore be regulated depending upon which A-type cyclin they bind and CDK1/cyclin A1 might be preferred in vivo.     

Design and synthesis of potent and selective pyridazin-4(1H)-one-based PDE10A inhibitors interacting with Tyr683 in the PDE10A selectivity pocket

Utilizing structure-based drug design techniques, we designed and synthesized phosphodiesterase 10A (PDE10A) inhibitors based on pyridazin-4(1H)-one. These compounds can interact with Tyr683 in the PDE10A selectivity pocket. Pyridazin-4(1H)-one derivative 1 was linked with a benzimidazole group through an alkyl spacer to interact with the OH of Tyr683 and fill the PDE10A selectivity pocket. After optimizing the linker length, we identified 1-(cyclopropylmethyl)-5-[3-(1-methyl-1H-benzimidazol-2-yl)propoxy]-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (16f) as having highly potent PDE10A inhibitory activity (IC₅₀ = 0.76 nM) and perfect selectivity against other PDEs (>13,000-fold, IC₅₀ = >10,000 nM). The crystal structure of 16f bound to PDE10A revealed that the benzimidazole moiety was located deep within the PDE10A selectivity pocket and interacted with Tyr683. Additionally, a bidentate interaction existed between the 5-alkoxypyridazin-4(1H)-one moiety and the conserved Gln716 present in all PDEs.     

Rotational relaxation time of concanavalin A bound to the surface membrane of normal and malignant transformed cells

Fluorescence polarization was used to determine the rotational relaxation time of fluorescein conjugated concanavalin A bound to the surface membrane of normal and malignant cells. The cells used were normal lymphocytes and malignant lymphoma cells, as examples of cells that are in suspension in vivo, and normal and simian virus 40-transformed fibroblasts, as examples of cells that form a solid tissue. The relaxation time of F-concanavalin A² in phosphate-buffered saline, pH 7.2, at 24 °C was 58 nseconds. Under the same conditions, the relaxation times of F-concanavalin A bound to cells were: 70 nseconds for normal lymphocytes, 160 nseconds for malignant lymphoma cells, 120 nseconds for normal fibroblasts and 73 nseconds for simian virus 40-transformed fibroblasts. When cells were treated with trypsin or glutaraldehyde before adding F-concanavalin A, trypsin treatment produced a decrease, whereas glutaraldehyde fixation produced an increase of these values. Inhibition of cap formation of concanavalin A binding sites on normal lymphocytes by treating the cells with sodium azide did not change the rotational relaxation time of concanavalin A bound to the cells. These results indicate that the carbohydrate-containing structures on the cells that bind concanavalin A are mobile. In cells that are in suspension in vivo, malignant transformation is associated with reduction in mobility of these sites. However, in cells that form a solid tissue, malignant transformation is associated with an increase in mobility of these sites. Determination of the rotational relaxation time of fluorescent probes bound to specific sites on cell membranes can thus be used to quantitate receptor mobility in relation to cell behaviour.     

Regulation of fetuin A gene expression in the neonatal pig liver

Fetuin A (also known as α2-Heremans–Schmid glycoprotein) is a protein primarily expressed by the liver and secreted into the blood. Previous studies have suggested that plasma concentrations of fetuin A are elevated with impaired growth rate in swine. The present study was designed to examine the relationship of porcine fetuin A with growth rate in the pig and to also elucidate the regulation of fetuin A expression by examining the hormonal and cytokine regulation of fetuin A mRNA abundance in hepatocytes prepared from suckling piglets. Quantitative real-time PCR assay was used to quantify the number of fetuin A mRNA molecules/molecule cyclophilin mRNA. Total RNA was isolated from liver of three different groups of pigs to assess changes in mRNA abundance of fetuin A: normal piglets at day 1, day 7 day 21 or 6 months of age (n=6 for each age); runt and control piglets at day 1 of age (n=4); slow growing and normal growing piglets at 21 days of age (n=8). Following birth, fetuin A gene expression increased from day 1 and 7 of age (P<0.05), and then declined at 21 days of age (P<0.05), with a much greater decline to 6 months of age (P<0.01). Fetuin A mRNA abundance was higher in runt pigs v. their normal birth weight littermates (P<0.05). Similarly, fetuin A gene expression was higher in livers of pigs that were born at a normal weight but that grew much slower than littermates with the same birth weight (P<0.05). Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h to permit examination of the influences of hormones, cytokines and redox modifiers on fetuin A mRNA abundance. Fetuin A gene expression was enhanced by glucagon, T3 and resveratrol (P<0.05). Growth hormone, cytokines (interleukin6, tumor necrosis factor-α) and antioxidants (N-acetylcysteine, quercertin) reduced fetuin A mRNA abundance (P<0.05). A role for fetuin A in postnatal development is suggested by the differences in fetuin A mRNA abundance between runt piglets or slow growing piglets and their normal growing sized littermates. The hepatocyte experiments suggest multiple hormones and cytokines may contribute to the regulation of fetuin A during early growth of the pig.     

Design and synthesis of novel benzimidazole derivatives as phosphodiesterase 10A inhibitors with reduced CYP1A2 inhibition

A novel class of phosphodiesterase 10A (PDE10A) inhibitors with reduced CYP1A2 inhibition were designed and synthesized starting from 2-{[(1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (1). Introduction of an isopropyl group at the 2-position and a methoxy group at the 5-position of the benzimidazole ring of lead compound 1 resulted in the identification of 2-{[(2-isopropyl-5-methoxy-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (25b), which exhibited potent PDE10A inhibitory activity with reduced CYP1A2 inhibitory activity compared to compound 1.     

Isolation of two new phenolic compounds from the fruit of Chaenomeles speciosa (Sweet) Nakai

Phytochemical research on the anti-inflammatory activities of Chaenomeles speciosa (Sweet) Nakai (Rosaceae) to investigate the main components of 10% ethanol fraction of the crude extract of C. speciosa fruit in an attempt to find bioactive compounds or new compounds from this medicinal plant. The phytochemical investigation succeeded in isolating two new phenolic compounds, specpolyphenol A (1) and specphenoside A (2), together with three known phenyl glycosides (3–5) from the fraction. The structures of the new compounds were deduced from comprehensive spectroscopic analyses including IR, EI-MS, ¹H NMR, ¹³C NMR, DEPT, COSY, HMBC and HMQC. The structures of the three known compounds 3, 4 and 5 were identified by comparison of their spectral data with those reported in the literature.     

Mapping and tissue mRNA expression analysis of the pig solute carrier 27A (SLC27A) multigene family

Solute-carrier family 27A molecules are integral transmembrane proteins that play a fundamental role in the uptake of long-chain fatty acids into mammalian cells. Our goal was to characterize this multigene family in pigs. Chromosomal location of the six porcine SLC27A genes was determined by radiation hybrid mapping and indicated that the six genes map to six different chromosomal locations. Moreover, we analyzed SLC27A mRNA expression in six pig tissues by quantitative RT-PCR. While SLC27A1, SLC27A3 and SLC27A4 were expressed in most, if not all, analyzed tissues, SLC27A2, SLC27A5 and SLC27A6 were predominantly expressed in the liver. In general, pig and human SLC27A mRNA expression profiles were remarkably concordant, although important differences were observed for SLC27A1 and SLC27A6 mRNAs. Discrepancies between mRNA expression profiles have been observed even in closely related primate species, and they might reflect the acquisition of regulatory changes promoting evolutionary adaptation.     

Preparative isolation of bio-markers from the leaf exudate of Aloe ferox (“aloe bitters”) by high performance counter-current chromatography

One of the most crucial factors determining the safety and efficacy of any herbal medicine or natural product-based formulation is the quality of the raw material. The absence of readily available bio-markers (standards) is one of the hurdles which need to be overcome to develop robust and effective quality control protocols.Aloe ferox Mill. is a most coveted ethnomedicinally import plant indigenous to South Africa. A. ferox has been used since ancient times in folk medicine and recently it has gained popularity as an ingredient in cosmetic formulations and food supplements. This study aimed to develop a superior method for the isolation of bio-markers from “aloe bitters” (exudate) obtained from A. ferox.For separation by HPCCC the solvent system comprising of EtOAc/n-BuOH/H₂O (3.5:1.5:5, v/v/v) was used in reversed phase mode. By this method, and only in one run, eight bio-markers were separated and isolated on semi-preparative scale including aloesin, aloeresin C, aloeresin A, 5-hydroxyaloin, aloin B, aloinoside B, aloin A and aloinoside A. The isolation of bio-active molecules from A. ferox (Cape aloes) is presented to illustrate the efficiency and advantages of high performance counter-current chromatography (HPCCC).     

KDM1A and KDM3A promote tumor growth by upregulating cell cycle-associated genes in pancreatic cancer

Pancreatic cancer is a highly malignant cancer of the pancreas with a very poor prognosis. Methylation of histone lysine residues is essential for regulating cancer physiology and pathophysiology, mediated by a set of methyltransferases (KMTs) and demethylases (KDMs). This study surveyed the expression of methylation regulators functioning at lysine 9 of histone 3 (H3K9) in pancreatic lesions and explored the underlying mechanisms. We analyzed KDM1A and KDM3A expression in clinical samples by immunohistochemical staining and searching the TCGA PAAD program and GEO datasets. Next, we identified the variation in tumor growth in vitro and in vivo after knockdown of KDM1A or KDM3A and explored the downstream regulators of KDM1A and KDM3A via RNA-seq, and gain- and loss-of-function assays. Eleven H3K9 methylation regulators were highly expressed in pancreatic cancer, and only KDM1A and KDM3A expression positively correlated with the clinicopathological characteristics in pancreatic cancer. High expression of KDM1A or KDM3A positively correlated with pathological grade, lymphatic metastasis, invasion, and clinical stage. Kaplan–Meier analysis indicated that a higher level of KDM1A or KDM3A led to a shorter survival period. Knockdown of KDM1A or KDM3A led to markedly impaired tumor growth in vitro and in vivo. Mechanistically, CCNA2, a cell cycle-associated gene was partially responsible for KDM1A knockdown-mediated effect and CDK6, also a cell cycle-associated gene was partially responsible for KDM3A knockdown-mediated effect on pancreatic cancer cells. Our study demonstrates that KDM1A and KDM3A are highly expressed in pancreatic cancer and are intimately correlated with clinicopathological factors and prognosis. The mechanism of action of KDM1A or KDM3A was both linked to the regulation of cell cycle-associated genes, such as CCNA2 or CDK6, respectively, by an H3K9-dependent pathway.     

Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents

A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19–C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.     

Sesquiterpenes from Russula sardonia

Three new sesquiterpenes, furanether A, furosardonin A and sardonialactone A (7-hydroxyblennin A), together with lactaral, vellerolactone, two known furan alcohols, lactarorufin A and cerevisterol, were isolated from Russula sardonia. Structures and stereochemistry were elucidated by spectral data and correlation to known compounds by chemical transformations.     

Sesquiterpene lactones from Chrysanthemum coronarium

The flower heads of Chrysanthemum coronarium afforded a new sesquiterpene lactone, dihydrocumambrin A, in addition to the known cumambrin A.     

Clonal overgrowth of esophageal smooth muscle cells in diffuse leiomyomatosis-Alport syndrome caused by partial deletion in COL4A5 and COL4A6 genes

This is a study of a patient who manifests all of the features of a diffuse leiomyomatosis-Alport syndrome (DL-ATS), and her two-year-old son who has already been diagnosed with Alport syndrome. Fourteen years ago, the patient underwent a partial esophageal resection followed by a replacement with jejunum. Recently, she underwent a surgical resection of the esophagus due to esophageal dysfunction. Genetic analyses of COL4A5 and COL4A6 on the X-chromosome were efficiently performed using the genomic DNA of her son. We have identified a novel deletion of 194-kb in length, encompassing COL4A5-COL4A6 promoters as well as nearly the entire large intron 1 of COL4A5 and intron 2 of COL4A6. To uncover the relationship of the esophagus-specific occurrence of the tumor and the expression of those genes, immunohistochemical analyses of type IV collagen α chains were conducted in the non-affected individuals. The esophageal smooth muscle-specific expression of α5(IV) and α6(IV) chains in the gastrointestinal tract was observed. Moreover, CAG repeat analysis of the androgen receptor gene and an immunohistochemical analysis in the leiomyoma revealed clonal overgrowth of the cells which received X-inactivation on the non-affected allele. These results may suggest that the dominant effect was caused by the partial deletion of the esophageal smooth muscle-specific genes, COL4A5 and COL4A6.     

3种七叶树属植物叶片气体交换特征和叶绿素荧光特性比较

在自然条件下,测定了中华七叶树（Aesculus chinensis）、黄花七叶树（A. octandra）和大花七叶树（A. hybrida）植物叶片的气体交换参数和叶绿素荧光参数,并对其进行比较。结果表明,3种植物的光补偿点差异较大,其中中华七叶树最低,为12.53 μmol·m^-2·s^-1、明显低于其它2个种（分别为36.11和46.41 μmol·m^-2·s^-1）;3种植物的光饱和点也存在较大差异,其中中华七叶树为1 475 μmol·m^-2·s^-1;明显高于其它2个种（分别为1 366.67和1 025 μmol·m^-2·s^-1）;3种植物的最大净光合速率同样存在显著性差异,其中中华七叶树为9.47 μmol CO₂·m^-2·s^-1,显著高于其它2个种（分别为5.91和2.30 μmol CO₂·m^-2·s^-1）,说明了中华七叶树具有较强的光合能力。中华七叶树的表观电子传递速率（ETR）为55.800,分别是黄花七叶树和大花七叶树的1.33、1.44倍;中华七叶树的PSⅡ总的光化学量子产额（Yield）为0.470,分别是黄花七叶树和大花七叶树的1.21、1.15倍;中华七叶树的光化学淬灭（qP）为0.975,分别是黄花七叶树和大花七叶树的1.10、1.10倍。3项荧光指标在不同树种之间差异性达显著水平,说明中华七叶树具有较高的电子传递活性和光能转化效率。     

Chemical relationships between firs of Japan and Taiwan

Cortical essential oil from Abies sachalinensis, A. veitchii, A. mariesii, A. firma, A. homolepis, and A. Kawakamii was analyzed for its mono and sesquiterpenoid constituents. Qualitatively the essential oils were similar to those from the New World firs. Using percent composition of monoterpenoid hydrocarbons, it was possible to completely separate most of the mentioned taxa from each other. A method was developed for calculation of similarity coefficients and construction of taxonomic structure on the basis of biosynthetic relationships between individual components of cortical oleoresin. The resulting chemosystematic dendrograms reflected similarity in material flow through various biosynthetic intermediates and paralleled in many features the morphological similarity relationships between the species studied.     

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.