The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape

Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic beta-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.     

Immunization of cultured juvenile rockfish Sebastes schlegeli against Microcotyle sebastis (Monogenea)

To determine whether immunization with Microcotyle sebastis antigen could induce protection against the parasite's establishment, naive juvenile rockfish were immunized by injection or immersion with whole worm antigen of M. sebastis. The infestation intensities of immunized groups following a challenge (2 wk after boosting) with 5000 M. sebastis eyed-eggs were significantly lower than those of control groups, when determined 7 wk postinfection. The fish in the groups boosted with M. sebastis antigen showed stronger protection than unboosted groups. The control group injected with FCA only showed a significantly smaller number of worms than the control group, which was immersed in PBS containing seawater. The results strongly suggest that both specific and nonspecific immune factors participate in the protection of rockfish against M. sebastis establishment.     

A new method for the study of chylomicron kinetics in vivo

Our understanding of the metabolism of chylomicrons, the lipoprotein that transports dietary fat from the intestine to peripheral tissues, is incomplete. The present studies were conducted to determine whether a labeled intravenous lipid emulsion could be used to estimate chylomicron triglyceride (TG) rate of appearance (R(a)) and thereby quantify the rate of intestinal fat absorption. After an overnight fast, healthy volunteers (n = 6) sipped a (3)H-labeled drink over 6.5 h at a rate of 175 mg fat. kg(-1). h(-1). Beginning at hour 5, an HPLC-purified, (14)C-labeled lipid emulsion was infused intravenously for 90 min. During the study, plasma total and chylomicron TG concentrations increased from 100 +/- 21 to 237 +/- 40 mg/dl and from undetectable to steady-state levels of 35 +/- 13 mg/dl, respectively. After a minor correction for VLDL contamination, tracer-determined chylomicron TG R(a) was 175 +/- 30 mg. kg(-1). h(-1), equal to the presumed ingestion rate. In summary, a radiolabeled intravenous lipid emulsion is able to accurately estimate chylomicron TG R(a) and therefore can be used to measure in vivo fat absorption rates.     

Toward an energy function for the contact map representation of proteins

We analyzed several energy functions for predicting the native state of proteins from an energy minimization procedure. We derived the parameters of a given energy function by imposing the basic requirement that the energy of the native conformation of a protein is lower than that of any conformation chosen from a set of decoys. Our work is motivated by a recent result which proved that the simple pairwise contact approximation of the energy is insufficient to satisfy simultaneously such a basic requirement for all the proteins in a database. Here, we investigate the reasons of such negative results and show how to improve the predictive power of methods based on energy minimization. We generated decoys by gapless threading, and we derive energy parameters by perceptron learning. We first considered hydrophobic contributions to the energy, defined in several ways, and showed that the additional hydrophobic terms enlarge slightly the number of proteins that can be stabilized together. Next, we performed various modifications of the pairwise energy term. We introduced (1) a distinction between inter-residue contacts on the surface and in the core of a protein and (2) a simple distance-dependent pairwise interaction in which a two-tier definition of contact replaces the original (single-tier) one. Our results suggest that a detailed treatment of the pairwise potential is likely to be more relevant than the consideration of other forces.     

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Inhibition of fungal cell wall synthesizing enzymes by trans-cinnamaldehyde

This study examined the inhibitory effects of trans-cinnamaldehyde (CA), an aromatic aldehyde derived from Cinnamomi Cortex, on Saccharomyces cerevisiae cell wall synthesizing enzymes in vitro. This compound was found to be a noncompetitive inhibitor of beta-(1,3)-glucan synthase and a mixed inhibitor of chitin synthase 1 with 50% inhibitory concentrations (IC50) of 0.84 and 1.44 mM, respectively. Chitin synthases 2 and 3 were less sensitive than chitin synthase 1 to CA. CA can be useful as a model compound of cell wall inhibitors for the development of effective antifungal agents.     

Nuclear magnetic resonance solution conformation of alpha-conotoxin AuIB, an alpha(3)beta(4) subtype-selective neuronal nicotinic acetylcholine receptor antagonist

The neuronal nicotinic acetylcholine receptors constitute a highly diverse group, with subtypes consisting of pentameric combinations of alpha and beta subunits. alpha-Conotoxins are a homologous series of small peptides that antagonize these receptors. We present the three-dimensional solution structure of alpha-conotoxin AuIB, the first 15-residue alpha-conotoxin known to selectively block the alpha(3)beta(4) nicotinic acetylcholine receptor subtype. The pairwise backbone and heavy-atom root mean square deviation for an ensemble of 20 structures are 0.269 and 0.720 A, respectively. The overall fold of alpha-conotoxin AuIB closely resembles that of the alpha4/7 subfamily alpha-conotoxins. However, the absence of Tyr(15), normally present in other alpha4/7 members, results in tight bending of the backbone at the C terminus and effectively renders Asp(14) to assume the spatial location of Tyr(15) present in other neuronal alpha4/7 alpha-conotoxins. Structural comparison of alpha-conotoxin AuIB with the alpha(3)beta(2) subtype-specific alpha-conotoxin MII shows different electrostatic surface charge distributions, which may be important in differential receptor subtype recognition.     

Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3

betaig-h3 is a transforming growth factor-beta-inducible cell adhesion molecule that has four characteristic homologous repeated domains. We made recombinant betaig-h3 proteins, which were highly active in mediating human corneal epithelial (HCE) cell adhesion and spreading. The 2nd and the 4th repeated domains were sufficient to mediate HCE cell adhesion. A sequence analysis showed that aspartic acid (Asp) and isoleucine (Ile) of the 2nd and the 4th domains are highly conserved in many fasciclin 1 homologous (fas-1) domains. Substitution mutational study identified these two amino acids are essential for cell adhesion. Synthetic peptides containing Asp and Ile, NKDIL and EPDIM derived from the 2nd and the 4th domains, respectively, almost completely blocked cell adhesion mediated by not only wild type betaig-h3 but also each of the 2nd and the 4th domains. These peptides alone were fully active in mediating cell adhesion. In addition, we demonstrated the functional receptor for betaig-h3 is alpha(3)beta(1) integrin. These results, therefore, establish the essential motifs within the 2nd and the 4th domains of betaig-h3, which interact with alpha(3)beta(1) integrin to mediate HCE cell adhesion to betaig-h3 and suggest that other proteins containing Asp-Ile in their fas-1 domains could possibly function as cell adhesion molecules.