Cytoplasmic Regulation of the Movement of E-Cadherin on the Free Cell Surface as Studied by Optical Tweezers and Single Particle Tracking: Corralling and Tethering by the Membrane Skeleton

The translational movement of E-cadherin, a calcium-dependent cell–cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and α-catenin without its NH₂-terminal half. These cadherins were labeled with 40-nm colloidal gold or 210-nm latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell–cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 μm by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (D_micro) of 1.2 × 10⁻¹⁰ and 2.1 × 10⁻¹⁰ cm²/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 μm² for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average D_micro for Fusion measured by SPT was small (0.2 × 10⁻¹⁰ cm²/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/μm, respectively, indicating a difference in the skeletal structures that produce these two effects.     

Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from Pseudomonas chlororaphis

The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P. chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu²⁺ and Sn⁴⁺, respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.     

High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.

Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.     

Taxonomic notes on Japanese species of the Nomada furva species group (Hymenoptera: Apidae)

The furva species group of the genus Nomada in Japan is taxonomically revised and the following three species are recognized: N. okubira Tsuneki, 1973, stat. nov., N. pulawskii Tsuneki, 1973, and N. taicho Tsuneki, 1973. Nomada okubira is raised to species rank, and N. etigonis Tsuneki, 1986, is synonymized under N. okubira. Detailed illustrations of male terminalia are provided for N. okubira and N. taicho. A key to the Japanese species is provided.     

Stable Patterns of Allelic Diversity at the Merozoite Surface Protein-1 Locus of Plasmodium falciparum in Clinical Isolates from Southern Vietnam

ABSTRACT The extent of allelic diversity at the Merozoite Surface Protein-1 locus of Plasmodium falciparum (PfMSP-1) was examined in isolates collected from symptomatic patients living in a mesoendemic area in southern Vietnam. The variable blocks 2, 4 and 10 were typed by polymerase chain reaction and 24 PfMSP-1 gene types were defined as unique combinations of allelic types detected in each variable block. Nineteen PfMSP-1 gene types were identified and 182 parasite populations were fully typed among 102 isolates. Forty-eight (47%) patients harbored more than one typed parasite population, and one patient had at least eight genetically distinct subpopulations. As previously shown in the same endemic area, recombination between blocks 4 and 10 was significantly less frequent than expected from random assortment of allelic types. The distribution of PfMSP-1 gene types, however, did not differ significantly from that observed in isolates collected in the same area 17-24 mo before the present study. Furthermore, the prevalence of the most common gene types and the average number of different gene types harbored by the same host did not decrease with age. This argues against the prominence of frequency-dependent immune selection of PfMSP-1 polymorphisms in this parasite population.     

Erythrocles microceps, a new emmelichthyid fish from kochi, japan

A new emmelichthyid,Erythrocles microceps, is described from 15 specimens collected in Mimase Fish Market and Tosa Bay, Kochi Prefecture, Japan. It differs from its most similar congener,E. acarina, in having a greater number of lateral line scales (70–72 vs. 62–67 inacarina), more elongate body (body depth 23.8–25.9% SL vs. 27–29% SL), and smaller head (head) length 26.8–28.4% SL vs. 34–36% SL).     

Selection of light or darkness, locomotor, and stridulatory activities in the cricket, Gryllus bimaculatus Degeer (Orthoptera: Gryllidae)

Male crickets, Gryllus bimaculatus Degeer, turned lights on or off in a chamber by a seesaw device: (1) during a 12 h, and (2) during a 24 h day. The crickets in (1) and the last-instar nymph in (2) turned the lights on and off at irregular intervals and duration. The selection rate for darkness was greater than that for light by an average exceeding 80%. The locomotor activity of the nymph in (1) was arrhythmic. In (1) the adults stridulated and were active in continuous darkness during the 12 h, while, in contrast in (2) they turned on the light and stridulated without switching the light off. The locomotor and stridulatory activities of the adult crickets in (2) were free-running. These activities resulted in a free-running rhythm of selection for light or darkness in (2). Under the conditions of the present experiments, the circadian pacemaker functioned in the same way in light and dark cycles as in constant light conditions.     

Cloning of the Genes That Control Formation of the Fimbrial Colonization Factor Antigen III (CFA/III) from an Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strain 260-1 produces colonization factor antigen III and heat-labile enterotoxin. A 55-kb plasmid controlling the expression of the colonization factor antigen was isolated from this strain after it was labeled with ampicillin resistance transposon, Tn3. When this plasmid was introduced into E. coli K-12 strains, it induced the formation of pili that were morphologically and immunologically identical to those on the surface of 260-1 cells, as examined by electron microscopic observation and with the specific antiserum. The physical map of the plasmid was constructed, and the 17.4-kb region was found to be responsible for the expression of the pili.