Localization of UDP-GalNAc:NeuAc alpha 2,3Gal-R beta 1,4(GalNAc to Gal)N-acetylgalactosaminyltransferase in human stomach. Enzymatic synthesis of a fundic gland-specific ganglioside and GM2.

A glycolipid detected in human gastric mucosa with anti-GM2 monoclonal antibody was characterized to be GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc beta 1-3Gal 1-4Glc-Cer (NGM-1), which was lost in gastric cancer tissue with complementary increase of GM2 sharing the same terminal carbohydrate structure as NGM-1 (Dohi, T., Ohta, S., Hanai, N., Yamaguchi, K., and Oshima, M. (1990) J. Biol. Chem. 265, 7880-7885). The study on differential expression of NGM-1 in gastric fundic mucosa, pyloric mucosa, gastric cancer, and various other tissues indicated that NGM-1 existed specifically in fundic mucosa. The content of GM3 and sialylparagloboside (SPG), which are the substrates for the synthesis of GM2 and NGM-1, respectively, were not significantly different in these tissues. Therefore, the presence of two kinds of beta 1,4GalNAc transferases having different substrate specificity was considered to be critical for the expression of NGM-1 and GM2. The activity of beta 1,4GalNAc transferase which synthesizes GM2 or NGM-1 was determined by detecting the products with specific monoclonal antibodies. The activity of beta 1,4GalNAc transfer to SPG was high in fundic mucosa, while it was absent in pyloric mucosa or cancer. On the other hand, the increased activity of beta 1,4GalNAc transfer to GM3 was observed in cancer tissues and cancer cell lines which were rich in GM2. Our conclusion is that the limited expression of NGM-1 in fundic mucosa and the increase of GM2 in cancer are attributed to two types of beta 1,4GalNAc transferases localized in each region with different substrate specificity; the one in fundic mucosa transfers GalNAc to SPG but not to GM3, and the other one enhanced in cancer transfers GalNAc to GM3 but not to SPG.     

Novel technique for preprocessing high dimensional time-course data from DNA microarray: mathematical model-based clustering

MOTIVATION: Classifying genes into clusters depending on their expression profiles is one of the most important analysis techniques for microarray data. Because temporal gene expression profiles are indicative of the dynamic functional properties of genes, the application of clustering analysis to time-course data allows the more precise division of genes into functional classes. Conventional clustering methods treat the sampling data at each time point as data obtained under different experimental conditions without considering the continuity of time-course data between time periods t and t+1. Here, we propose a method designated mathematical model-based clustering (MMBC). RESULTS: The proposed method, designated MMBC, was applied to artificial data and time-course data obtained using Saccharomyces cerevisiae. Our method is able to divide data into clusters more accurately and coherently than conventional clustering methods. Furthermore, MMBC is more tolerant to noise than conventional clustering methods. AVAILABILITY: Software is available upon request. CONTACT: taizo@brs.kyushu-u.ac.jp.     

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.     

A new sigillid ostracod from submarine caves of the Ryukyu Islands, Japan

A new troglobitic sigillid genus, Kasella , is found in the submarine caves of the Ryukyu Islands. Sigillids are primitive podocopid ostracods having ancient characters such as unequal valves and many small adductor muscle scars. Kasella is possibly derived from the sigillid Cardobairdia , a deep-sea muddy bottom inhabitant, or its relatives. Submarine caves are characterized as both 'safe places', where biological factors such as competition and predation are reduced, and as 'crypts', representing dark and oligotrophic environments. It is likely that Kasella has successfully colonized hard substrates in caves, particularly exploiting the crevices on cave walls and ceilings. Kasella may have adapted to this cryptic habitat in response to strong cytheroidean competition and predation by, for example, gastropods that do not survive well in dark and oligotrophic submarine caves. In adapting to an oligotrophic cave environment, Kasella has undergone significant divergence in carapace morphology, possibly accompanying functional and behavioural changes.     

Embryonic senescence and laminopathies in a progeroid zebrafish model

Background

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist.

Principal Findings

We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape.

Conclusion

We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.     

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β-sheet composed of 22 β-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.     

Dielectric analysis of mitochondria isolated from rat liver. I. Swollen mitoplasts as simulated by a single-shell model

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer, L. and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589-601, and ibid. 7, 603-612) is presented. The suspensions of 'mitoplasts' prepared from rat liver mitochondria by a hyposmotic (10 mM KCl) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1-100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after scrutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297-305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 microF/cm2, (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6-0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.     

Redox-dependent structural ambivalence of the cytoplasmic domain in the inner ear-specific cadherin 23 isoform

Cadherin 23 (Cdh23), an essential factor in inner ear mechano-electric transduction, exists in two alternatively spliced forms, Cdh23(+68) and Cdh23(−68), depending on the presence and absence of exon 68. Cdh23(+68) is inner ear-specific. The exon 68-corresponding region confers an α-helical configuration upon the cytoplasmic domain (Cy) and includes a cysteine residue, Cys³²⁴⁰. We demonstrate here that Cy(+68) as well as the transmembrane (TM) plus Cy(+68) region is present in two different forms in transfected cells, reduced and non-reduced, the latter existing in more compact configuration than the former. The observed characteristic of Cy(+68) was completely abolished by Cys³²⁴⁰Ala substitution. Treatment of TMCy(+68)-transfected cells with diethyl maleate, a glutathione depleting reagent, resulted in conversion of the non-reduced to the reduced form of TMCy(+68), suggesting glutathione to be a Cys³²⁴⁰-binding partner. Multiple alignment of mammalian Cdh23Cy sequences indicated the occurrence of conformation-inducible Cys in Cdh23Cy of mammals, but not lower vertebrates. The implications of Cys-dependent structural ambivalence of Cdh23 in inner ear mechanosensation are discussed.     

Phylogeography of ten native herbaceous species in the temperate region of Japan: implication for the establishment of seed transfer zones for revegetation materials

Revegetation using native species requires the development of seed transfer zones that capture genetic distinctiveness and adaptive potentials while avoiding potential maladaptation and genetic contamination by exotic genotypes. Delineation based on phylogeographic information has recently been used to establish seed transfer zones; however, only a few herbaceous species that are suitable for revegetation have been investigated in the temperate regions of Japan. We investigated the phylogeography of non-coding regions of chloroplast DNA of ten native species in the temperate regions of Japan. Although no species showed clear-cut geographical distributions of the 2–14 haplotypes identified, spatially constrained Bayesian clustering showed two clusters in five species (Calamagrostis epigejos, Eragrostis ferruginea, Imperata cylindrica, Microstegium japonicum, and Microstegium vimineum) but not for others. Posterior modes of clusters for I. cylindrica and M. vimineum showed delineations at Chubu (the middle of Honshu Island), which divide the study region into northeastern and southwestern regions, indicating that these species had recovered from glacial refugia. Posterior mode of cluster for E. ferruginea showed that one consists of a coastal zone along the Pacific Ocean side of western Japan, while the other consists of the remaining area, indicating range expansion from south coast to north. Delineation of C. epigejos and M. japonicum were unclear. The mixed results indicated that establishing seed transfer zones for herbaceous species in Japan will require phylogeographical studies on a wide range of species that may be suitable for revegetation.