A pertussis toxin-sensitive GTP-binding protein plays a role in the G0-G1 transition of rat hepatocytes following establishment in primary culture

Acute spontaneous c-myc gene expression and sustained increase of a GTP-binding protein(s) (G-protein) which is sensitive to islet-activating protein (IAP), pertussis toxin, occurred early during primary culture of adult rat hepatocytes. Following these earlier events, DNA synthesis was demonstrated in response to EGF and insulin. Addition of IAP immediately after plating of primary cultures inhibited c-myc expression and the hormone-induced DNA synthesis. Addition at 24 h or later following cell inoculation, however, produced only weak effects on DNA synthesis, even though the IAP-sensitive G-proteins were completely inactivated. We conclude that the IAP-sensitive G-protein(s) plays a role in the earlier process(es) of the G0-G1 transition, which is essential for the initiation of growth factor-dependent DNA synthesis.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies

Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.     

Crystallographic Insights into the Pore Structures and Mechanisms of the EutL and EutM Shell Proteins of the Ethanolamine-Utilizing Microcompartment of Escherichia coli

The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.Bacterial microcompartments are subcellular organelles that are found in many prokaryotic organisms (10, 32). In contrast to the lipidic vesicles of many eukaryotic cells, these enclosures are entirely composed of proteins. Recent imaging by electron microscopy revealed capsid-like particles obeying 2-, 3- and 5-fold symmetries that suggest icosahedral symmetry (4, 13, 27). Shell proteins are thought to form a tightly sealed membrane structure that separates the lumen from the cytosol. Similar to the lipidic membranes of vesicles, these proteinaceous membranes have been suggested to provide a selectively permeable solute barrier, wherein specific pores maintain an optimal biochemical environment for the catabolic reactions inside (25).The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) enables some bacteria to survive on ethanolamine as the sole source for carbon, nitrogen, and energy (25). It is encoded by a 17-gene-containing operon, and homologues of its genes have been identified in Escherichia coli, Salmonella enterica serovar Typhimurium, Mycobacterium tuberculosis, and Clostridium kluyveri among other prokaryotic pathogens (22). Largely based on sequence comparisons, the compartment''s outer shell was proposed to be composed of five different shell proteins: Eut-K, -L, -M, -N, and -S, all of which are fairly small proteins that typically consist of about 100 amino acids. Only EutL is about twice the size, with 216 amino acids as a result of two tandemly duplicated shell protein domains (26).To date, little is known about the composition, architecture, and function of bacterial microcompartments. Recent structural investigations of BMC particles and individual shell proteins, however, have contributed greatly to a basic understanding of BMC architecture. Electron microscopy, for example, has revealed polyhedral shell structures that are composed of a thin layer of proteins. Intriguingly, crystallizations revealed that some shell proteins also assemble into tightly packed two-dimensional arrays that may resemble the facets of the compartments (28). Within an array, these proteins typically assembled into hexamers or trimers (in the case of tandem domain proteins) that exhibited a distinct hexagonal shape. As this geometry was suggested to be of fundamental importance to the microcompartment architecture, we will here refer to it as a “tile” or “tile structure.” While it has not yet been proven directly that the assembly of proteins in the crystals is identical to that of the BMC, their almost seamless two-dimensional packing has been suggested to be of physiological relevance as it could provide an efficient barrier to prevent leakage of toxic by-products into the cytoplasm (4, 25). Overall, however, it is not understood how the various shell proteins assemble to form the polyhedral structure while maintaining an efficiently tight seal. In particular, the interactions among the shell proteins and their arrangements within facets, edges, and vertices have remained elusive.In the study presented here, we demonstrate for the first time that the shell protein EutM is also able to form tightly packed two-dimensional arrays. With two independently determined crystal structures, we show that its protein array closely resembled that of EutL and other carboxysomal proteins. As a result, we hypothesize that this assembly represents a physiologically relevant form. Both crystal forms also revealed the C-terminal tail of the protein, which is proposed to serve as a potential interaction site with other factors.Furthermore, we show that the pore structure of EutL can be triggered to open upon exposure to specific solutes. A first structure of EutL was previously determined in our laboratory, and it revealed three water-filled pores per tile (26). Interestingly, its structure consisted of two tandemly repeated shell protein domains, which assembled into an almost perfectly shaped hexagonal structure. This architectural feature was recently also found in shell proteins of other microcompartments (11, 20). Each of the pores of an EutL tile was coated with acidic residues, which indicated a possible pathway for positively charged molecules such as ethanolamine. Inspection of the structure also suggested specific metal binding sites on its surface. In order to verify this idea, we performed systematic soaking studies of the crystals with selected divalent metals. Surprisingly, we found that zinc ions bound to the protein specifically not at the suspected sites but at different sites that caused a dramatic opening of a central pore. This unprecedented observation of a specifically triggered pore opening is consistent with another previous observation (30) and may point to a mechanism for regulation of permeability.     

CpG/CpNpG motifs in the coding region are preferred sites for mutagenesis in the breast cancer susceptibility genes

The range of BRCA1/BRCA2 gene mutations is diverse and the mechanism accounting for this heterogeneity is obscure. To gain insight into the endogenous mutational mechanisms involved, we evaluated the association of specific sequences (i.e. CpG/CpNpG motifs, homonucleotides, short repeats) and mutations within the genes. We classified 1337 published mutations in BRCA1 (1765 BRCA2 mutations) for each specific sequence, and employed computer simulation combined with mathematical calculations to estimate the true underlying tendency of mutation occurrence. Interestingly, we found no mutational bias to homonucleotides and repeats in deletions/insertions and substitutions but striking bias to CpG/CpNpG in substitutions in both genes. This suggests that methylation-dependent DNA alterations would be a major mechanism for mutagenesis.     

Using model substrates to study the dependence of focal adhesion formation on the affinity of integrin-ligand complexes

The adhesion of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix and the clustering of these receptors into focal adhesion complexes. This paper examines the effect of one mechanistic variable, ligand affinity, on the assembly of focal adhesions (FAs) in order to gain mechanistic insight into this process. This study uses self-assembled monolayers of alkanethiolates on gold as a substrate to present either a linear or cyclic Arg-Gly-Asp peptide at identical densities. Inhibition assays showed that the immobilized cyclic RGD is a higher affinity ligand than linear RGD. 3T3 Swiss fibroblasts attached to substrates presenting the cyclic peptide at twice the rate they attached to substrates presenting the linear peptide. Quantitation of focal adhesions revealed that cells on cyclic RGD had twice the number of FAs as did cells on linear RGD and that these focal adhesions were on average smaller. These findings show that affinity affects the assembly of integrins into focal adhesions and support a model based on competing rates of nucleation and growth of FAs to explain the change in distribution of FAs with ligand affinity. This study is important because it provides a model system that is well-suited for biophysical studies of integrin-mediated cell adhesion and reveals insight into one mechanism utilized by cells to perceive environmental changes.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

Isolation of a novel thermophilic fungus <Emphasis Type="Italic">Chaetomium</Emphasis> sp. nov. MS-017 and description of its palm-oil mill fiber-decomposing properties

Palm-oil mill fiber (POMF) is a fibrous, natural hard material discharged in enormous amounts from palm-oil mills in tropical plantations; therefore, research to find microorganisms that decompose POMF was conducted. As the result of screening, a new thermophilic fungus, Chaetomium sp. nov. MS-017, exhibiting rapid growth on POMF was isolated from rotted wood. Based on partial characterization of the decomposition of POMF, it was shown that MS-017 preferentially assimilates polysaccharides, especially hemicelluloses such as xylan. A preliminary composting study indicated that MS-017 produced 855 g of decomposed product from 1,000 g of intact POMF in 12 days under optimized solid-culture conditions. The decomposition rate of POMF was 23% (w/w), and the cell yield calculated from consumed POMF was as high as 36% (w/w). These results indicate that MS-017 has a very high potential to decompose POMF and that it is suitable for economical production of compost to recycle by-product biomass from oil-palm plantations.     

Complete mitochondrial genome of the Baikal teal Anas formosa (Aves, Anseriformes, Anatidae)

The Baikal teal Anas formosa (Aves, Anseriformes, Anatidae) is classified as "Vulnerable" on the IUCN Red List. Here, whole mitochondrial genome of A. formosa was amplified and sequenced. The total length of the Baikal teal mitochondrial genome is 16,594?bp, which consists of 13 protein-coding, 2 rRNA, 22 tRNA genes and 1 control region. The characteristics of the mitochondrial genomes were analyzed and discussed in detail.