Kisspeptin Effect on Endothelial Monocyte Activating Polypeptide II (EMAP-II)-Associated Lymphocyte Cell Death and Metastases in Colorectal Cancer Patients

Kisspeptin is an antimetastatic agent in some cancers that has also been associated with lymphoid cell apoptosis, a phenomenon favoring metastases. Our aim was to determine the association of kisspeptin with lymphocyte apoptosis and the presence of metastases in colorectal cancer patients. Blood was drawn from 69 colon cancer patients and 20 healthy volunteers. Tissue specimens from healthy and pathological tissue were immunohistochemically analyzed for kisspeptin and endothelial monocyte activating polypeptide II (EMAP-II) expression. Blood EMAP-II and soluble Fas ligand (sFasL) levels were examined by an enzyme-linked immunosorbent assay method. The kisspeptin and EMAP-II expression and secretion levels in the DLD-1 and HT-29 colon cancer cell lines were examined by quantitative real-time polymerase chain reaction, Western analysis and enzyme-linked immunosorbent assay, whereas lymphocyte viability was assessed by flow cytometry. The effect of kisspeptin on the viability of colon cancer cells was examined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Exogenous, synthetic and naturally produced, kisspeptin induces through the G-protein-coupled receptor 54 (GPR54; also known as the kisspeptin receptor) the EMAP-II expression and secretion in colon cancer cell lines, inducing in vitro lymphocyte apoptosis, as verified by the use of an anti-EMAP-II antibody. These results were reversed with the use of kisspeptin inhibitors and by kisspeptin-silencing experiments. Tumor kisspeptin expression was associated with the tumor EMAP-II expression (p < 0.001). Elevated kisspeptin and EMAP-II expression in colon cancer tissues was associated with lack of metastases (p < 0.001) in colon cancer patients. These data indicate the antimetastatic effect of tumor-elevated kisspeptin in colon cancer patients that may be mediated by the effect of kisspeptin on EMAP-II expression in colon cancer tumors in patients with normal serum EMAP-II levels. These findings provide new insight into the role of kisspeptin in the context of metastases in colon cancer patients.     

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

A cellulose synthesis complex with a “rosette” shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the “hexamer of trimers” model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.Cellulose, the most abundant biopolymer on Earth, is composed of linear chains of β-1,4 linked d-Glc monomers with repeating structural units of the disaccharide cellobiose. Numerous cellulose polymers cocrystallize to form microfibrils, which provide mechanical strength and rigidity to plants. Its natural abundance makes it an attractive target for many industrial applications, including paper and pulping, construction, and textile manufacture. More recently, cellulose has been used for production of biofuels, such as ethanol (Ragauskas et al., 2006; Langan et al., 2014), and in the form of nanocellulose as a component in advanced composite materials (Reddy et al., 2013; Habibi, 2014). Cellulose microfibrils are synthesized by a large membrane-bound protein complex. In the land plants and charophycean algae, the cellulose synthesis complex (CSC) has a “rosette” shape (Mueller et al., 1976; Mueller and Brown, 1980b; Kimura et al., 1999), and the entire CSC has reported diameters between 24 to 30 nm (Lerouxel et al., 2006). This structural information was revealed by freeze-fracture transmission electron microscopy, showing six lobes in a hexagonal arrangement at the point where the transmembrane helices of multiple cellulose synthase proteins (CESAs) cross the plasma membrane. Recently KORRIGAN, a protein with cellulase activity, has also been implicated as an integral component of the CSC (Vain et al., 2014).Vascular plants produce several different CESA isoforms. For example, Arabidopsis (Arabidopsis thaliana) has 10 different isoforms with 64% to 98% sequence identity (Holland et al., 2000; Richmond, 2000; McFarlane et al., 2014). The different CESA isoforms play specific roles in cellulose synthesis during plant development. In Arabidopsis, CESA1, CESA3, and CESA6 are required for primary cell wall synthesis, while CESA4, CESA7, and CESA8 are required for secondary cell wall synthesis (Gardiner et al., 2003; Taylor et al., 2003; Persson et al., 2007). CESA2, CESA5, and CESA9 play roles in tissue-specific processes and are partially redundant with CESA6, whereas CESA10 is closely related to AtCESA1 but evidently has a minor role in plant development (Somerville, 2006). The absolute number of CESA proteins present in a CSC remains a subject of much speculation, largely because the stoichiometry of the cellulose microfibril remains unresolved (Cosgrove, 2014). The traditional representation of the microfibril has 36 cellulose chains, and based on this, one would expect that each lobe of the rosette CSC contains six CESA proteins responsible for the synthesis of six glucan chains for a total of 36 CESA proteins per rosette CSC (Herth, 1983; Perrin, 2001; Doblin et al., 2002). However, recent studies using different analytical techniques combined with computation report 18 to 24 cellulose chains per microfibril (Fernandes et al., 2011; Thomas et al., 2013; Oehme et al., 2015). A study of cellulose from mung bean (Vigna radiata) primary cell walls, using x-ray diffraction, solid-state NMR, and computational analysis, supports an 18-chain model for a cellulose microfibril (Newman et al., 2013). This implies that the CSC is composed of fewer than 36 CESA proteins or that not all of the proteins in a CSC are simultaneously active. Further, it has been recently reported that the stoichiometry of CESAs 1, 3, and 6 and CESAs 4, 7, and 8 in the primary and secondary cell walls, respectively, is 1:1:1 (Gonneau et al., 2014; Hill et al., 2014). Together, these reports suggest a rosette CSC composed of 18 CESA proteins with three CESAs per lobe as the most likely composition of a rosette CSC to account for an 18-chain cellulose microfibril (Newman et al., 2013; Gonneau et al., 2014; Hill et al., 2014). In addition, it should also be noted that 24 CESA proteins in a rosette CSC with four proteins per lobe is incompatible with a 1:1:1 CESA stoichiometry.Numerous efforts to isolate active CESA proteins directly from plants or by recombinant expression have not been successful, preventing a detailed structural analysis of CESA proteins or the mechanism of plant cellulose synthesis. In contrast, the recently reported crystal structure of cellulose synthase from Rhodoobacter sphaeroides (Morgan et al., 2013) clearly showed that only a single cellulose synthase polypeptide is required for glucan polymerization and also identified the conserved sequence motifs responsible for catalysis. In addition, based on the presence of an 18-residue glucan chain in the protein tunnel, a mechanism for cellulose synthesis and translocation across a cytoplasmic membrane was proposed that also addressed how the alternate d-Glc molecules are inverted during polymer synthesis (Morgan et al., 2013; Omadjela et al., 2013). However, this structure cannot provide insight into the formation of microfibrils from the cellulose chains synthesized by single polypeptides of CESA.The CESA proteins of land plants and their charophycean algal relatives are multidomain single polypeptide chains of approximately 1000 amino acids. They are predicted to have eight transmembrane helices and to have their N- and C-terminal regions facing the cytoplasm (Pear et al., 1996). Although they share sequence similarity with the bacterial counterpart, they also have unique structural features not found in the bacterial enzymes. The N-terminal domain contains a Zn-binding site that may play a role in oligomerization of CESA proteins (Kurek et al., 2002). The putative cytosolic domain, which is flanked by a two-helix N-terminal transmembrane domain and a six-helix C-terminal transmembrane domain (McFarlane et al., 2014; Slabaugh et al., 2014), has D, D, D, QxxRW motifs that are conserved substrate binding and catalytic residues in the glycosyltransferase-2 superfamily (Nagahashi et al., 1995; Pear et al., 1996; Saxena and Brown, 1997; Yoshida et al., 2000). This domain also has a plant-conserved region (P-CR) and a class-specific region (CSR) that are only found in CESAs that form rosette CSCs. Although the roles of these regions are unknown, they are proposed to be involved in regulatory functions, such as interactions with other proteins and oligomerization to form the rosette shape. In the Arabidopsis CESAs, the sequence identity within the P-CR regions is greater than 80%, while in CSR regions, it is only about 40%. A recent computational model of the cytosolic domain of cotton (Gossypium hirsutum) CESA1 provides the first detailed structural model of the catalytic domain of CESA (Sethaphong et al., 2013). This model structure aligns well with the crystal structure of the bacterial cellulose synthase, indicating that a common mechanism exists for cellulose synthesis in bacteria and plants and that CESAs within rosette CSCs contain a single active synthetic site. In addition, this model made it possible to test possible configurations for the assembly of CESA monomers into a functional rosette CSC (Newman et al., 2013; Sethaphong et al., 2013).Our understanding of the mechanism of cellulose biosynthesis in plants at the molecular level is hampered by the lack of an atomic level CESA model. To gain deeper insight into the structure and role of the catalytic domain of CESA in rosette formation, we carried out a structural characterization of the cytosolic domain of Arabidopsis CESA1, a protein that is essential for cellulose synthesis in the primary cell wall (Arioli et al., 1998). The recombinant protein was purified from Escherichia coli in a two-step process that allowed us to obtain low-resolution structural information about the monomeric and trimeric forms of the recombinant protein using small-angle scattering (SAS) techniques. This study provides the first experimental evidence to support the self-assembly of CESAs into a stable trimer complex, revealing the possible role of the catalytic domain in the formation of the rosette CSC. Comparison of the size of the catalytic domain trimer with dimensions of rosette CSCs obtained from TEM studies strongly supports the “hexamer of trimers” model for rosette CSCs. Computational analysis of the scattering data suggested configurations for how the monomers, including the plant-specific P-CR and CSR domains, may be arranged in the trimeric lobes of the rosette CSC. Knowledge of how CESA proteins assemble in the CSC will enable approaches for rational genetic manipulation of plant cell wall synthesis, which offers enormous opportunities to improve feedstocks for the production of sustainable fuels and chemicals.     

Photolabile opioid derivatives of D-Ala2-Leu5-enkephalin and their interactions with the opiate receptor

Photolabile derivatives of D-Ala2-Leu5-enkephalin were prepared by synthetic procedures in which a 2-nitro-4-azidophenyl group is linked to the terminal carboxyl group of the enkephalin by means of an ethylenediamine or ethylenediamine beta-alanine spacer. These peptides bind to opiate receptors with nanomolar affinities and inhibit electrically stimulated contractions of the mouse vas deferens and adenylate cyclase activity of NG108-15 neuroblastoma x glioma hybrid cell membranes. Both inhibitions are reversed by the opiate antagonist naloxone. Photolysis of the ligands bound to rat brain membranes results in the loss of approximately 50% of the receptor sites. This decrease in receptor number is blocked by naloxone and requires light. A photolabile [3H]enkephalin derivative labels an equivalent number of sites under similar irradiation conditions.     

Genic VERSUS Chromosomal Variation in Natural Populations of DROSOPHILA SUBOBSCURA

Gametic frequencies in one mainland and one island population of D. subobscura were obtained by means of extracting wild chromosomes and subsequently analyzing them for inversions and allozymes. The high degree of cytological heterogeneity which characterizes these populations is not reflected in the genetic data. Two cases of non-random association were observed among eighteen pair-wise comparisons involving gene alleles and inversions to which the locus is linked. In both cases exchange of alleles at the locus is completely suppressed by the inversions. Four cases of linkage disequilibrium were detected among eighteen pairs of loci; two of them could best be explained as transient associations generated by random drift. The results suggest that disequilibria among enzyme loci are not widespread in natural populations—Populations with a lower degree of chromosomal variation are genetically as variable as populations with a higher degree of chromosomal variation. This observation does not support the hypothesis that selection in marginal homokaryotypic populations is for specialized homozygous genotypes.     

The Genetics of DROSOPHILA SUBOBSCURA Populations. Xiv. Further Data on Linkage Disequilibria

Data coming from one natural population of D. subobscura, that of Crete, are presented in detail and examined for nonrandom associations of genes and gene arrangements. This population and four others previously studied are reanalyzed for the detection of higher than first-order interactions. Only first-order interactions are important and statistically significant, especially those concerning genes and inversions in which these genes are included. The paucity of linkage disequilibria detected is remarkable, and we argue that it does not depend on the methods of study, rather it is genuine. We further argue that most of the disequilibria detected are probably due to mechanisms based on epistatic selection.     

Opioid activities and structures of alpha-casein-derived exorphins

Exorphins, peptides with opioid activity, have previously been isolated from pepsin hydrolysates of alpha-casein [Zioudrou, C., Streaty, R. A., & Klee, W. A. (1979) J. Biol. Chem. 254, 2446-2449]. Analysis of these peptides shows that they correspond to the sequences 90-96, Arg-Tyr-Leu-Gly-Tyr-Leu-Glu, and 90-95, Arg-Tyr-Leu-Gly-Tyr-Leu, of alpha-casein. These peptides, as well as two of their analogues Tyr-Leu-Gly-Tyr-Leu-Glu (91-96) and Tyr-Leu-Gly-Tyr-Leu (91-95), have now been synthesized and characterized. Their opioid activity was examined by three different bioassays: (a) displacement of D-2-alanyl[tyrosyl-3,5-3H]enkephalin-(5-L-methioninamide) and [3H]dihydromorphine from rat brain membranes; (b) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells; (c) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens. The synthetic peptide of sequence 90-96 was the most potent opioid in all three bioassays and its potency was similar to that of the isolated alpha-casein exorphins. The synthetic peptides were totally resistant to hydrolysis by trypsin and homogenates of rat brain membranes, but were partially inactivated by chymotrypsin and subtilisin. The difference in opioid activity of alpha-casein exorphins may be related to differences in conformational flexibility observed by NMR spectroscopy.     

Differentiation of the vitellogenin proteins in species of the Drosophila obscura group

The three female specific vitellogenin proteins of eight Drosophila species of the obscura group were detected in discontinuous SDS-polyacrylamide gels. The molecular weights of these proteins were estimated. These three genetic markers represent an useful addition to the electrophoretic variation that has been used to construct phylogenies in the genus Drosophila.     

Electrophoretic patterns of chorion proteins and their relation to phylogeny of eleven Drosophila species of the melanogaster group

The electrophoretic patterns of chorion proteins coded by four chorion genes in eleven Drosophila species of the melanogaster group have been studied. We found that, in spite of the specific characteristics of this unique set of genes, the electrophoretic patterns are, in general, in accordance to the proposed phylogenies.     

A portrait of the “SCP/TAPS” proteins of eukaryotes — Developing a framework for fundamental research and biotechnological outcomes

A wide range of proteins belonging to the SCP/TAPS “family” has been described for various eukaryotic organisms, including plants and animals (vertebrates and invertebrates, such as helminths). Although SCP/TAPS proteins have been proposed to play key roles in a number of fundamental biological processes, such as host–pathogen interactions and defence mechanisms, there is a paucity of information on their genetic relationships, structures and functions, and there is no standardised nomenclature for these proteins. A detailed analysis of the relationships of members of the SCP/TAPS family of proteins, based on key protein signatures, could provide a foundation for investigating these areas. In this article, we review the current state of knowledge of key SCP/TAPS proteins of eukaryotes, with an emphasis on those from parasitic helminths, and undertake a comprehensive, systematic phylogenetic analysis of currently available full-length protein sequence data (considering characteristic protein signatures or motifs) to infer relationships and provide a framework (based on statistical support) for the naming of these proteins. This framework is intended to guide genomic and molecular biological explorations of key SCP/TAPS molecules associated with infectious diseases of plants and animals. In particular, fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new and innovative approaches for the control of parasitic diseases, with important biotechnological outcomes.