CRMP-2 is involved in kinesin-1-dependent transport of the Sra-1/WAVE1 complex and axon formation

A neuron has two types of highly polarized cell processes, the single axon and multiple dendrites. One of the fundamental questions of neurobiology is how neurons acquire such specific and polarized morphologies. During neuronal development, various actin-binding proteins regulate dynamics of actin cytoskeleton in the growth cones of developing axons. The regulation of actin cytoskeleton in the growth cones is thought to be involved in axon outgrowth and axon-dendrite specification. However, it is largely unknown which actin-binding proteins are involved in axon-dendrite specification and how they are transported into the developing axons. We have previously reported that collapsin response mediator protein 2 (CRMP-2) plays a critical role in axon outgrowth and axon-dendrite specification (N. Inagaki, K. Chihara, N. Arimura, C. Menager, Y. Kawano, N. Matsuo, T. Nishimura, M. Amano, and K. Kaibuchi, Nat. Neurosci. 4:781-782, 2001). Here, we found that CRMP-2 interacted with the specifically Rac1-associated protein 1 (Sra-1)/WASP family verprolin-homologous protein 1 (WAVE1) complex, which is a regulator of actin cytoskeleton. The knockdown of Sra-1 and WAVE1 by RNA interference canceled CRMP-2-induced axon outgrowth and multiple-axon formation in cultured hippocampal neurons. We also found that CRMP-2 interacted with the light chain of kinesin-1 and linked kinesin-1 to the Sra-1/WAVE1 complex. The knockdown of CRMP-2 and kinesin-1 delocalized Sra-1 and WAVE1 from the growth cones of axons. These results suggest that CRMP-2 transports the Sra-1/WAVE1 complex to axons in a kinesin-1-dependent manner and thereby regulates axon outgrowth and formation.     

Lambertellol C, a labile and novel biosynthetic congener of lambertellols A and B

Lambertellol C (1), a labile biosynthetic congener of lambertellols A and B, was isolated from a fermentation broth of Lambertella sp. 1346 based on the diagnostic isotope patterns in the mass spectrum of a highly labeled sample. The CD spectrum of 1 indicated that this lambertellol analogue exists as a racemate. The mechanism for this racemization is discussed.     

Optimization of isotope-labeling conditions for lambertellin based on isotope patterns observed by mass spectrometry

A method for the sensitive analysis of the incorporation level of labeled acetate was developed. This method allowed for the optimization of the conditions for lambertellin with up to 48% average incorporation of labeled acetate.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP31 and MIP32 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP31 and MIP32 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP31 and MIP32 contain four exons and three introns, and MIP31 and MIP32 shared the same intron/exon organization with that of human. The MIP31 and MIP32 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP32 gene was much lower than the Trsc-SCYA107 and MIP31 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP31 and MIP32 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP32 genes in PWBCs was observed at 1–12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP31 expression was observed at 3–12 h poststimulation only with PMA.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin

Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.     

Cloning and characterization of the homolog of mammalian lipopolysaccharide-binding protein and bactericidal permeability-increasing protein in rainbow trout Oncorhynchus mykiss

We cloned two cDNAs denoted as RT-LBP/BPI-1 and RT-LBP/BPI-2, respectively, which were derived from the mRNA of head kidney from rainbow trout. They showed structural homology with LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) in mammals. The full-length cDNA of RT-LBP/BPI-1 and RT-LBP/BPI-2 is 1666 and 1741 bp, respectively. Both cDNAs encoded 473 aa residues, including the amino acids conserved in mammalian LBP and BPI proteins that were assumed to be involved in LPS binding. The overall coding sequence of RT-LBP/BPI-1 has 33% amino acid homology to human LBP and 34% to human BPI, and RT-LBP/BPI-2 has 32% amino acid homology to human LBP and 33% to human BPI. Three-dimensional structure analysis by three-dimensional/one-dimensional (3D-1D) methods also demonstrated that RT-LBP/BPI-1 and RT-LBP/BPI-2 proteins showed significant similarity to human BPI, having a boomerang shape with N-terminal and C-terminal barrels. Phylogenetic analysis showed that the LBP and BPI genes seemed to be established after the divergence of mammals from teleosts. These results suggested that RT-LBP/BPI-1 and RT-LBP/BPI-2 may be a putative ortholog for mammalian LBP and/or BPI genes. This is the first study to identify the LBP family genes from nonmammalian vertebrates.     

Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients

Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the C-terminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.     

Detection of marine birnavirus in the Japanese pearl oyster Pinctada fucata and seawater from different depths

This study examines the seasonal changes of marine birnavirus (MABV) in seawater and the Japanese pearl oyster Pinctada fucata reared at different depths (2 and 15 m). Oysters and seawater were collected in 1998, and a 2-step PCR was carried out to detect MABV. Virus isolation was performed on the PCR-positive samples in the oyster. The detection rate of the MABV genome in the oyster was low during June, but increased after July at both 2 and 15 m depths. MABV was not isolated until after September, when isolation rates of 10 to 28.6% were recorded. The results suggest that growth of MABV in the oyster is similar at 2 and 15 m depth. In contrast, the MABV genome in seawater was present through the year at 15 m depth, but was not detected in summer at 2 m. This suggests that the virus is destroyed by UV and/or other factors at 2 m in summer, but is stable in deeper waters.