Molecular characterization of Bombyx mori cytoplasmic polyhedrosis virus genome segment 4

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Proteins of the CNR family are multiple receptors for Reelin

Layering and positioning of neurons require Reelin- and Src family-associated mammalian Disabled (mDab1). Cadherin-related neuronal receptor (CNR) genes are expressed in neurons of the cortical layer, but not in Cajal-Retzius cells expressing Reelin. This leads us to hypothesize that CNRs bound to Fyn of the Src family are receptors for Reelin. Herein we confirm the association and colocalization of CNR proteins with Reelin. This binding is blocked by CR-50 antibody against Reelin, as well as by monoclonal antibodies produced against CNRs. Both disturb the signaling pathway from Reelin to mDab1 and the positioning of cortical neurons in vitro. These results strongly suggest that the CNR family proteins are multiple Reelin receptors. In addition, differential conservation of the Reelin-binding domain among terrestrial vertebrates may be pertinent to the diversity or complexity of brains.     

Genomic sequence and organization of the family of CNR/Pcdhalpha genes in rat

CNR/Pcdhalpha family proteins are known as synaptic cadherins and Reelin receptors. Here we report the complete genomic sequence and organization of the rat CNR. The rat CNR cluster encodes 15 variable and 3 constant exons. The genomic organizations of the rat, mouse, and human CNR/Pcdhalpha are orthologous. The percentage identity of the coding regions between the rat and the mouse is 93.6% on average at the nucleic acid level, and between rat and human it is 82.8%. The rat CNRs (v1-v13) also contain an RGD motif in the extracellular cadherin 1 domains and cysteine repeats that are characteristic of the transmembrane and cytoplasmic domains of CNR proteins. The number of variable exons in the rat CNR cluster is identical to that of the human. The rat CNR cluster has one more variable exon than is found in laboratory mouse strains, because in the mouse a variable exon located between v7 and v8 is divided by the insertion of a retrotransposon. This exon is not disrupted in the rat, in which it is transcribed. By in silico analysis, CNR/Pcdhalpha was also mapped to rat chromosome 18, but the orientation was opposite for the mouse CNR/Pcdhalpha gene cluster. The relative expression profiles of the rat CNRs (v1-v13) show that all the CNRs are transcribed, but there are variations in the expression ratios among the CNRs.     

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.