A novel, brain-specific mouse drebrin: cDNA cloning, chromosomal mapping, genomic structure, expression, and functional characterization

Drebrin A, a major neuronal actin-binding protein, regulates the dendritic spine shapes of neurons. Here, we have cloned and characterized a novel mouse cDNA clone encoding a truncated form of drebrin A, named s-drebrin A. Analysis of the genomic organization of the mouse drebrin gene (Dbn1), which mapped to the central portion of chromosome 13, revealed that isoforms including s-drebrin A are generated by alternative splicing from a single drebrin gene. The s-drebrin A mRNA was expressed in the brain, but not in non-neuronal tissues. The s-drebrin A expression was barely detected in the embryonic brain, but was upregulated during postnatal development of the brain. Overexpression of GFP-tagged s-drebrin A in fibroblasts showed it to be associated with actin filaments and with changes in actin cytoskeleton organization. These findings suggest that s-drebrin A has a role in spine morphogenesis, possibly by competing the actin-binding activity with drebrin A.     

A human gene encoding a protein homologous to ribosomal protein L39 is normally expressed in the testis and derepressed in multiple cancer cells

We identified and characterized a gene encoding a protein that was 92% identical to human ribosomal protein L39. This gene was located on the long arm of chromosome 3, and was composed of three exons and two long introns. Analysis of mRNA expression in 16 types of normal human tissues showed that this gene was expressed specifically in the testis, in sharp contrast to the ubiquitous expression of the ribosomal protein L39 gene. Surprisingly, the new gene was expressed in 19 out of 24 human cancer samples of various tissue origins. When the new gene was expressed in the cell, a translated product was observed by immunofluorescence microscopy in the nucleus, especially strongly in the nucleolus, and in the cytoplasm. Association of this protein with the large subunit of cytoplasmic ribosomes was detected by polyacrylamide-agarose composite gel electrophoresis followed by immunodetection. These immunochemical data suggest a relationship between the new gene and the ribosome.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.     

Isolation and characterization of 39 microsatellite loci in the endangered Japanese loach Leptobotia curta

A microsatellite-enriched genomic library was obtained for the endangered Japanese loach Leptobotia curta, and 39 dinucleotide markers were successfully isolated and characterized. These markers had between one and nine alleles, with expected heterozygosity ranging from 0 to 0.839, in a population from the Lake Biwa-Yodo River system of Japan. Linkage equilibrium was observed in most loci, and only one locus showed significant deviation from Hardy-Weinberg equilibrium. These microsatellite markers will be useful for genetic diversity studies of wild and captive L. curta populations.     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.     

An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells

The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 μg 10⁻⁶ cells day⁻¹, one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-κB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation, was examined. Suppression of NF-κB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.     

Absorption, migration and kinetics in peripheral blood of orally administered ovalbumin in a mouse model

Intestinal absorption of food proteins is well known, whereas its physiological significance remains to be investigated. Various amounts (1, 10 and 50 mg) of ovalbumin were orally administered to mice and the blood kinetics were subsequently analyzed by two-site ELISA. The blood ovalbumin concentration consistently reached its maximum (7-90 ng/ml) about 20 min after the oral administration and then gradually decreased in a dose-dependent manner. Only intact (45 kDa) and truncated (40 kDa) ovalbumins were always detected in the blood independently of the administration site, intra-stomach or intra-intestine, while various fragments of the protein were observed in the gastrointestinal lumen after the oral administration. Recognition by a specific monoclonal antibody and an acidic shift of its pI value suggested that the 40-kDa truncated ovalbumin was produced by intracellular limited proteolysis at its C-terminus. Such stable absorption and blood kinetics of undigested ovalbumin in normal mice suggest some sort of physiological significance for the intestinal uptake of intact food proteins.     

Periodate-resistant carbohydrate epitopes recognized by IgG and IgE antibodies from some of the immunized mice and patients with allergy

The β(1,2)-xylose- and/or α(1,3)-fucose-containing cross-reactive carbohydrate determinants (CCDs) are present in various plant and insect N-glycans, and have been attracted as potential antigens in IgE-mediated allergies and immunologically undesired post-translational products on some recombinant therapeutic proteins. By using ELISA and immunoblotting, CCDs-specific IgG and IgE antibodies from some, but not all, of mice and humans were found to fully retain their binding activity after a typical periodate-treatment to CCDs, which did cause the CCDs’ antigenic activity to those from the other mice and rabbits to disappear almost completely. Furthermore, the mouse IgE recognizing the periodate-resistant CCDs induced the CCDs/IgE-dependent degranulation of rat basophilic RBL-2H3 cells. These findings indicate that in some cases CCDs include those dependent of the core trisaccharide more strongly than the terminal xylose and fucose, which might have been screened out in the CCDs analyses based on the loss of antibody-binding by the periodate-treatment.     

A mutation in the uvi4 gene promotes progression of endo-reduplication and confers increased tolerance towards ultraviolet B light

We have isolated and characterized a new ultraviolet B (UV-B)-resistant mutant, uvi4 (UV-B-insensitive 4), of Arabidopsis. The fresh weight (FW) of uvi4 plants grown under supplemental UV-B light was more than twice that of the wild-type. No significant difference was found in their ability to repair the UV-B-induced cyclobutane pyrimidine dimers, or in the amount of UV-B absorptive compounds, both of which are well-known factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. We found that the hypocotyl cells in uvi4 undergo one extra round of endo-reduplication. The uvi4 mutation also promoted the progression of endo-reduplication during leaf development. The UVI4 gene is expressed mainly in actively dividing cells. In the leaves of P(UVI4)::GUS plants, the GUS signal disappeared in basipetal fashion as the leaf developed. The total leaf blade area was not different between uvi4 and the wild-type through leaf development, while the average cell area in the adaxial epidermis was considerably larger in uvi4, suggesting that the uvi4 leaves have fewer but larger epidermal cells. These results suggest that UVI4 is necessary for the maintenance of the mitotic state, and the loss of UVI4 function stimulated endo-reduplication. Tetraploid Arabidopsis was hyper-resistant to UV-B compared to diploid Arabidopsis, suggesting that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4 mutant.