On hypervariability at the reactive center of proteolytic enzymes and their inhibitors

The pattern of synonymous and nonsynonymous substitutions at the reactive center of proteases (kallikrein) and their inhibitors (₁-antitrypsin and serpin) was examined. In the case of ₁-antitrypsin, the proportion of different nonsynonymous sites exceeds that of different synonymous sites at the reactive center for sequence pairs of recent duplication. The result indicates that the positive selection has operated after duplication to increase functional diversity. In the cases of kallikrein, serpin, and remote sequence pairs of ₁-antitrypsin, the proportion of different synonymous sites at the reactive center exceeds that of different synonymous sites at the remaining region. The result indicates that gene conversion followed by natural selection is working. On the whole, it is concluded that hypervariability of amino acids at the reactive center is generated by an interaction among natural selection, random genetic drift, point mutation, and gene conversion. Gene duplication may provide potential for them to interact.     

Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thaliana.

cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.     

Behavior of mitochondria and their nuclei during amoebae cell proliferation inPhysarum polycephalum

Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - BrdU 5-bromodeoxyuridine - FITC fluorescein isothiocyanate     

Further Examples of Evolution by Gene Duplication Revealed through DNA Sequence Comparisons

To test the theory that evolution by gene duplication occurs as a result of positive Darwinian selection that accompanies the acceleration of mutant substitutions, DNA sequences of recent duplication were analyzed by estimating the numbers of synonymous and nonsynonymous substitutions. For the troponin C family, at the period of differentiation of the fast and slow isoforms, amino acid substitutions were shown to have been accelerated relative to synonymous substitutions. Comparison of the first exon of α-actin genes revealed that amino acid substitutions were accelerated when the smooth muscle, skeletal and cardiac isoforms differentiated. Analysis of members of the heat shock protein 70 gene family of mammals indicates that heat shock responsive genes including duplicated copies are evolving rapidly, contrary to the cognitive genes which have been evolutionarily conservative. For the α(1)-antitrypsin reactive center, the acceleration of amino acid substitution has been found for gene pairs of recent duplication.     

Crystallization and Analyses of Crystals of Various Chemotypes of R-Form Lipopolysaccharides from Salmonella Spp.

Various chemotypes (Re, Rd₂, Rd₁P^–, Rd₁, RcP^–, Rc, Rb₃, Rb₂, Rb₁, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl₂, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Å. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.     

Pattern of Nucleotide Substitutions in Growth Hormone-Prolactin Gene Family: A Paradigm for Evolution by Gene Duplication

The growth hormone-prolactin gene family in mammals is an interesting example of evolution by gene duplication. Divergence among members of duplicated gene families and among species was examined by using reported gene sequences of growth hormone, prolactin and their receptors. Sequence divergence among species was found to show a general tendency in which a generation-time effect is pronounced for synonymous substitutions but not so for nonsynonymous substitutions. Divergence among duplicated genes is characterized by the relatively high rate of nonsynonymous substitutions, i.e., the rate is close to that of synonymous ones. In view of the stage- and tissue-specific expression of duplicated genes, some of the amino acid substitutions among duplicated genes is likely to be caused by positive Darwinian selection.     

A synergistic effect of glutathione-depletion and elicitation on the production of 6-methoxymellein in carrot cells

The effects of buthionine sulfoximine (BSO) and yeast glucan elicitor (YE) on the production of 6-methoxymellein (6-MM) and generation of H₂O₂ in suspension-cultured carrot cells were examined. Administration of BSO and YE together affected the cells synergistically to lead to an enhanced production of 6-MM. These data indicate the significance of formation and decay of active oxygen species as a second signal of elicitation in triggering the biosynthesis of the phytoalexin.Abbreviations BSO buthionine sulfoximine - MDA malondialdehyde - 6-MM 6-methoxymellein - YE yeast glucan elicitor     

A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human and 1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.     

Age-Associated Increase of CD5+ B Cells in the Liver of Autoimmune (NZB × NZW) F1 Mice

The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5⁺ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB × NZW) F₁ mice. An age-associated increase of CD5⁺ B cells was demonstrated in the liver of these mice. Although CD5⁺ B cells (i.e., CD5⁺IgM⁺ and CD5⁺B220⁺) constituted a minor population of hepatic mononuclear cells (MNC) (<5%) when mice were young (8 weeks), a large population of CD5⁺ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age-dependent increase of CD5⁺ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5⁺ B cells was the same as that of CD5⁺ B cells in the peritoneal cavity and spleen, showing dull-CD5, bright-IgM and dull-B220. High levels of CD5⁺ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5-day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM- and IgG-autoantibodies against double-stranded DNA and single-stranded DNA, despite the increased proportion of CD5⁺ B cells. On the other hand, peritoneal exudate cells produced only IgM-, but not IgG-, autoantibodies, whereas splenic cells were able to produce both IgM- and IgG-autoantibodies. These results suggest that the liver might support the generation of the most primitive CD5⁺ B cells in these mice and that such generation increases as a function of age, probably resulting in the onset of autoimmune disease.