Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer

The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells, serve as ligands for a cell adhesion molecule of the selectin family, E-selectin, which is expressed on vascular endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelium and thus contribute to hematogenous metastasis of cancer. The initial adhesion mediated by these molecules triggers activation of integrin molecules through the action of several cytokines and leads to the extravasation of cancer cells. Cancer cells also produce humoral factors that facilitate E-selectin expression on endothelial cells. The degree of expression of the carbohydrate ligands at the surface of cancer cells is well correlated with the frequency of hematogenous metastasis and prognostic outcome of patients with cancers. The alteration of glycosyltransferase activities that leads to the enhanced expression of these carbohydrate ligands on cancer cell surface are currently being investigated. This revised version was published online in November 2006 with corrections to the Cover Date.     

Structural and energetic parameters of Ca2+ binding to peptides and proteins

The characteristics of Ca²⁺-binding sites and of the structural reorganization induced by Ca²⁺-binding in storage proteins and ion carriers are being studied as models for molecular mechanisms in Ca²⁺ channels and in Ca²⁺-dependent modulatory proteins. A first step in the study was the development of energy parameters for Ca²⁺ compatible with those in the CHARMM package of computer simulation software. Such parameters were obtained from an analytical fit to the potential surface for [(Ca)(OCH₂)₄]²⁺ calculated with an ab initio molecular orbital method. The resulting parametrization was tested for the hexapeptide cyclo-(Pro-Gly)₃, and a 75 residue long calcium binding protein from bovine intestine (ICaBP). The geometrical parameters calculated for the hexapeptide and its 2:1 complex with Ca²⁺ were in good agreement with experimental data from crystallography and nmr. Similarly, the structure of ICaBP optimized with CHARMM using the new Ca²⁺ parameters showed good agreement with the x-ray structure both in the local structures of the calcium-binding sites and in the overall shape of the protein.     

Using Acoustics to Determine Eelgrass Bed Distribution and to Assess the Seasonal Variation of Ecosystem Service

Eelgrass beds are an important source of primary production in coastal ecosystems. Understanding seasonal variation in the abundance and distribution of eelgrass is important for conservation, and the objectives of this study were to 1) monitor seasonal variation in eelgrass beds using an acoustic monitoring method (Quantitative echo sounder) and 2) broadly quantify the carbon circulation function. We obtained acoustic data of eelgrass beds in coastal areas north and east of Ikunojima Island. Surveys were conducted nine times over the 3-year period from 2011 to 2013 in order to monitor seasonal variation. Acoustic data were obtained and used to estimate the spatial distribution of eelgrass by geostatistical methods. To determine supporting services, we determined carbon sink and carbon fixation by eelgrass beds using data from the National Research Institute of Fisheries and Environment of Inland Sea (2011). The height and distribution of eelgrass beds were at a maximum in May and at a minimum in November of each year. Distribution trends were different between the north and east areas. Supporting services showed the same patterns throughout the year. The area of distribution was considered to be coincident with the life history of eelgrass. Distribution differed by area and changed yearly due to the effects of bottom characteristics and wind direction. Quantifying the supporting services of eelgrass beds was shown to be useful for managing the conservation of coastal ecosystems.     

Self-organization of the heartbeat as coordination among ventricular myocardial cells through mechano-electrical feedback

To elucidate the mechanism of the self-organizing control of heartbeats, models that include the electrical and mechanical processes of heartbeat are proposed. As the mutual interaction between the two processes, mechano-electrical feedback (MEF), mediated by stretch-activated ionic channels, is considered. Analyses of the models show that coordination of beats among myocardial cells is established by MEF even in the absence of electrical coupling. The coordination of heartbeats is found to show in-phase synchronization under normal conditions, while it is out of phase or irregular under mechanically abnormal conditions. It is concluded that coupling by MEF is important for the self-organization of heartbeats. Received: 4 December 1997 / Accepted in revised form: 29 July 1998     

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.     

Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C

Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.     

Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.