Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication

Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft-associated molecules such as glycosylphosphatidylinositol (GPI)-anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP-1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI-anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB-dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.     

Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.     

Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner

Motility of nerve growth cones (GCs) is regulated by region-specific activities of cell adhesion molecules (CAMs). CAM activities could be modified by their localization to detergent-resistant membranes (DRMs), specialized microdomains enriched in signaling molecules. This paper deals with a question of whether DRMs are involved in GC migration stimulated by three CAMs; L1, N-cadherin (Ncad), and beta1 integrin. We demonstrate that L1 and Ncad are present in DRMs, whereas beta1 integrin is exclusively detected in non-DRMs of neurons and that localization of L1 and Ncad to DRMs is developmentally regulated. GC migration mediated by L1 and Ncad but not by beta1 integrin is inhibited after DRM disruption by micro-scale chromophore-assisted laser inactivation (micro-CALI) of GM1 gangliosides or by pharmacological treatments that deplete cellular cholesterol or sphingolipids, essential components for DRMs. Characteristic morphology of GCs induced by L1 and Ncad is also affected by micro-CALI-mediated DRM disruption. Micro-CALI within the peripheral domain of GCs, or even within smaller areas such as the filopodia and the lamellipodia, is sufficient to impair their migration. However, micro-CALI within the central domain does not affect GC migration. These results demonstrate the region-specific involvement of DRMs in CAM-dependent GC behavior.     

Melina: motif extraction from promoter regions of potentially co-regulated genes

'Melina' assists users to compare the results of four public softwares for DNA motif extraction in order to both confirm the reliability of each finding and avoid missing important motifs. It is also useful to optimize the sensitivity of software with a series of different parameter settings. AVAILABILITY: Melina is available at http://www.hgc.ims.u-tokyo.ac.jp/Melina/.     

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) mRNAs in pheochromocytomas. Methods: The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. Results: mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH (p=0.0018) and PNMT (p=0.05) mRNA expression, as well as epinephrine (p=0.0342) levels in 16 intra-adrenal pheochromocytomas. Conclusion: Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.     

Small open reading frames in 5' untranslated regions of mRnas

Using the 5'-end sequence data from 'oligo-capped' cDNAs, we generated a representative full-length cDNA dataset for 4870 RefSeq entries, and analyzed the 5' untranslated region (UTR) of these genes. To our surprise, about half of the 4870 genes had an upstream ATG before the ATG that starts the longest open reading frame (ORF), suggesting that about half of them have small ORFs in their 5' UTR of average length of 31 amino acids. They require attention for further analysis to identify their biological role.     

Serological relationships among genotypic variants of betanodavirus

Betanodaviruses, the causative agents of viral nervous necrosis or viral encephalopathy and retinopathy, are divided into 4 genotypes based on the coat protein gene (RNA2). In the present study, serological relationships among betanodavirus genotypic variants were examined by virus neutralization tests using rabbit antisera raised against purified virions of strains representative of each genotype. All 20 isolates examined shared epitopes for neutralizing, but they fell into 3 major serotypes (A, B, C). This sero-grouping is in part consistent with their genotypes, i.e. Serotype A for striped jack nervous necrosis virus (SJNNV) genotype, Serotype B for tiger puffer nervous necrosis virus (TPNNV) genotype, and Serotype C for both redspotted grouper nervous necrosis virus (RGNNV) and barfin flounder nervous necrosis virus (BFNNV) genotypes. The serological relatedness between RGNNV and BFNNV genotypes may result from their relatively higher similarity in RNA2 sequences. In neutralization tests using antisera of kelp grouper Epinephelus moara, which were raised against recombinant coat proteins representing each genotype, anti-SJNNV and anti-TPNNV sera neutralized only the homologous strain, and anti-RGNNV and anti-BFNNV sera reacted with both RGNNV and BFNNV strains. The present serological findings will be important in investigating the infectivity and host-specificity of betanodaviruses and in developing vaccines for the disease.