Toshiro Hamamoto

Differential sublocalization of the dynamin-related protein OPA1 isoforms in mitochondria.

OPA1 is a cause gene for autosomal dominant optic atrophy and possesses eight alternative splicing variants. Here, we identified two isoforms of OPA1 proteins in HeLa cells and examined their submitochondrial localization and complex formations. RT-PCR shows that HeLa cells mainly express isoforms 7 and 1 of OPA1. Since the third cleavage site is mainly utilized in HeLa cells, the predicted molecular masses of their processed proteins are consistent with the 93- and 88-kDa proteins. Biochemical examinations indicate that both of the OPA1 isoforms are present in the intermembrane space. Submitochondrial fractionation by sucrose density-gradient centrifugation shows that the 88-kDa protein predominantly associates with the mitochondrial outer membrane, on the contrary, the 93-kDa protein associates with the inner membrane. Gel filtration analysis indicates that they compose the different molecular mass complexes in mitochondria. These differences between two isoforms of OPA1 would suggest their crucial role involved in the mitochondrial membrane formation.

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.

Isoliquiritigenin suppresses pulmonary metastasis of mouse renal cell carcinoma

Isoliquiritigenin is a chalcone isolated from licorice and shallots. The ability of isoliquiritigenin to suppress metastasis was examined in a pulmonary metastasis model of mouse renal cell carcinoma. Isoliquiritigenin significantly reduced pulmonary metastasis, without any weight loss or leukocytopenia. Isoliquiritigenin suppressed in vitro proliferation of carcinoma cells, potentiated nitric oxide production by lipopolysaccharide-stimulated macrophages, and facilitated cytotoxicity of splenic lymphocytes in vitro. These findings suggest activation of macrophages, activation of cytotoxicity of lymphocytes, and direct cytotoxicity as possible mechanisms of metastasis suppression by isoliquiritigenin. In addition, isoliquiritigenin prevented severe leukocytopenia caused by administration of 5-fluorouracil.

Net ATP synthesis in H+-ATPase macroliposomes by an external electric field

Application of electric pulses (1000 V/cm, 20 m sec duration) to macroliposomes containing pure stable H+-ATPase (F0·F1) resulted in synthesis of ATP. Microliposomes containing F0·F1 showed very little ATP synthesis under the same conditions. The amount of ATP synthesized was increased by increasing the number of electric pulses applied and decreased by addition of either an uncoupler or an energy transfer inhibitor.

Molecular cloning and expression of chick embryo Galβ1,4GlcNAcα2,6‐sialyltransferase

DNA clones encoding beta-galactoside alpha 2,6-sialyltransferase have been isolated from chick embryonic cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open-reading frame coding for 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region and a large C-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Gal beta 1,4GlcNAc in glycoproteins.

Two step single primer mediated polymerase chain reaction. Application to cloning of putative mouse, β-galactoside α2,6-sialyltransferase cDNA

Abstract Using the 2 step single primer mediated polymerase chain reaction(PCR), mouse β-galactoside (α2,6-sialyltransferase cDNA was cloned. Single primer mediated PCR is a method to amplify a particular DNA fragment beyond its known sequence region. It employs only one primer for the reaction. Compared to other PCR methods to amplify an adjacent sequence of known DNA fragment, this method requires no enzymatic manipulation on template DNA and is applicable to a template on long DNA fragment. First, a short DNA fragment of the enzyme was obtained from mouse cDNA by the usual PCR method using degenerate primers synthesized according to a relatively conserved region in rat and human β-galactoside (α2,6-sialyltransferase. Four primers were synthesized based on this sequence, then 2 step single primer mediated PCR were performed to obtain 5′ and 3′ flanking sequences of this short fragment resulting in 1.0 kb and 1.3 kb fragments being amplified respectively. The integrity of the two fragments was confirmed by an additional PCR using primers synthesized according to the joined sequence, which contained 1.2kb complete putative mouse β-galactoside α2,6-sialyltransferase coding region. The result showed that the specificity and consequently applicability of the single primer mediated PCR for amplifying a particular DNA fragment beyond known sequence region was remarkably improved by the successive 2nd reaction.

Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase γ subunit (F1γ) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492–8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998)Biochem. Biophys. Res. Commun. 251, 603–608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F1γ gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F1γ wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F1γ Pu-del and F1γ Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F1γ Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F1γ Pu-rich minigene mice revealed that the F1γ Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F1γ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

Molecular cloning and expression of chick Gal, β1,3GalNAc α2,3-sialyltransferase

Abstract A cDNA clone encoding chick Gal β1,3GalNAc α2,3-sialyltransferase (ST3Gal I) was isolated from a chick embryo brain cDNA library. The cDNA sequence included an open reading frame coding for 342 amino acids, and the deduced amino acid sequence showed 64% identity with that of the mouse enzyme. Northern blot analysis of chick embryos revealed that the ST3Gal I gene was expressed in early embryonic stages. The identity of the enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminal part including the cytoplasmic tail and signal anchor domain was replaced with an immunoglobulin signal peptide sequence. This enzyme expressed in COS-7 cells exhibited transferase activity similar to that of mouse ST3Gal I.

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F0F1) is driven by an electrochemical potential of H+ (δΜH+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The subunit composition of F1 is α3β3γδε. The active α3β3 oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F1 (TF1). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the βDELSEED sequence via γδε-F0. In fact, cross-linking of βDELSEED to γ stopped the ATP-driven rotation of γ in the center of α3β3. The torque of the rotation is estimated to be 420 pN·å from the δΜH+ and H+-current through F0F1. The angular velocity (Ω) of γ is the rate-limiting step, because δΜH+ increased theVmax of H+ current through F0, but not theKm(ATP). The rotational unit of F0 (=ab2c10) is π/5, while that in α3β3 is 2π/3. This difference is overcome by an analog-digital conversion via elasticity around βDELSEED with a threshold to release ATP. The αβ distance at the C site is about 9.6 å (2,8-diN3-ATP), and tight Mg-ATP binding in α3β3γ was shown by ESR. The rotational relaxation of TF1 is too rapid (Φ=100 nsec), but the rate of AT(D)P-induced conformational change of α3β3 measured with a synchrotron is close to Ω. The ATP bound between the P-loop and βE188 is released by the shift of βDELSEED from γRGL. Considering the viscosity resistance and inertia of the free rotor (γ-c), there may be a stator containing OSCP (=δ of TF1) and F0-d to hold free rotation of α3β3.

Cytochrome c-551 of the thermophilic bacterium PS3, DNA sequence and analysis of the mature cytochrome

The structural gene for cytochrome c-551 was isolated from genomic DNA of the thermophilic bacterium PS3. The amino acid sequence of cytochrome c-551 as deduced from the DNA sequence consists of 111 amino acid residues and contains one heme c-binding site (-CASCH-) located approximately in the middle of the polypeptide. The N-terminus of isolated cytochrome c-551 was blocked, but treatment with Rhizopus lipase and molecular weight measurement of the mature and lipase-treated forms by ion spray mass spectroscopy suggest that the mature c-551 may have 93 or 94 amino acid residues with a diacylated glycerol-cysteine at the N-terminal region. The first 17 or 18 amino acid residues in the N-terminal region of the nascent polypeptide, rich in hydrophobic and basic amino acid residues, may be a signal peptide to translocate the major portion of cytochrome c-551 to the extracellular surface and to be processed. Similarity of amino acid sequence of this protein is discussed in relation to other c-type cytochromes of bacilli as well as bacterial small cytochromes c such as Pseudomonas aeruginosa cytochrome c-551 and cytochrome c6 of cyanobacteria.