Biomedical subjects

K Sutoh

Publications and source records attributed to K Sutoh.

At least 37 records · Page 2Linked to original sources

PubMed1998

Dictyostelium TRFA homologous to yeast Ssn6 is required for normal growth and early development.

The TPR (tetratricopeptide repeat) family became widespread during evolution, having been found from bacteria to mammals. By means of restriction enzyme-mediated integration, we have identified a Dictyostelium gene (trfA) highly homologous to a Saccharomyces cerevisiae gene encoding a TPR protein, Ssn6 (Cyc8), which functions as a global transcriptional repressor for diverse genes. The deduced amino acid sequence of the Dictyostelium gene product, TRFA, contains 10 consecutive TPR units as well as Gln repeats, Asn repeats, and a region rich in Glu, Lys, Ser, and Thr. The sequences of some of the 10 TPR units in TRFA are more than 70% identical to the corresponding units in Ssn6. The trfA- cells produced smooth plaques on a bacterial lawn and failed to aggregate normally when starved on a plain agar plate. Individual trfA- cells also failed to correctly respond to cAMP, although the adenylyl cyclase of trfA- cells was expressed upon starvation and activated by stimulation with cAMP as in the wild-type cells. When cultured in a rich medium in suspension, they grew more slowly and stopped growing at a lower density than the wild-type cells. Furthermore, they divided into cells of various sizes and tended to be much smaller than the wild-type cells. These pleiotropic defects of the trfA- cells suggest the possibility that Dictyostelium TRFA may regulate the transcription of diverse genes required for normal growth and early development.

Amino Acid Sequence↗

PubMed1998

Mutational analysis of the switch II loop of Dictyostelium myosin II.

A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the "switch II loop" of the myosin family is DIXGFE. In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the Dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions, whereas the D454A, G457A, F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis steps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.

Actins↗

PubMed1998

High incidence of esophageal cancer in esophageal achalasia by the oral administration of N-amyl-N-methylnitrosamine and its prevention by nicardipine hydrochloride in mice.

Esophageal achalasia (EA) is a rare disease in man and animals and there are many discussions on its higher risk of esophageal cancer. N-Amyl-N-methylnitrosamine (AMN) which specifically induces esophageal tumors in mice and rats was given to three mutant mouse strains, i.e. 101/N, STX/Le and BXH-8, which develop a high incidence of EA. The incidence of EA in 101/N, STX/Le, BXH-8 and normal C57BL/6J mice was 38.5% (110/286), 30.1% (43/143), 91.8% (190/207) and 0% (0/167), respectively. The average numbers of AMN-induced esophageal tumors in EA(+) were significantly higher than those of EA(-) in all of the 101/N, STX/Le and BXH-8 mice. Furthermore, significantly larger size tumors and invasive squamous cell carcinomas were found in EA(+) mice than in EA(-) mice. These results indicate the higher sensitivity of EA for both tumor induction and promotion, possibly due to the longer retention of AMN. In fact, relaxation of the lower esophagus by a smooth muscle relaxing calcium-channel blocker, nicardipine hydrochloride, significantly prevented the induction of esophageal tumors.

Administration, Oral↗

PubMed1998

A novel Dictyostelium discoideum gene required for cAMP-dependent cell aggregation.

Using a method of random insertional mutagenesis called REMI (restriction enzyme-mediated integration), we isolated two mutant strains of Dictyostelium discoideum with a defect in cAMP-dependent cell aggregation. On bacterial lawns, both of the cells formed large and smooth plaques. When starved in a non-nutrient medium, they became elongated and extended pseudopods very frequently like starved wild type cells. However, they never formed streams toward an aggregation center. Genomic DNA fragments flanking the sites of insertion of the REMI tag were rescued from the mutant cells. The fragments contained one common open reading frame encoding a protein of 1148 amino acid residues. The protein's sequence is homologous to those of two hypothetical proteins of S. cerevisiae and S. pombe.

Amino Acid Sequence↗

PubMed1998

Functional characterization of Dictyostelium discoideum mutant myosins equivalent to human familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in beta-myosin heavy chain or other various sarcomeric proteins. To elucidate the functional impact of FHC mutations in myosin heavy chain, we generated Dictyostelium discoideum myosin II mutants equivalent to human FHC mutations by site-directed mutagenesis, and characterized their molecular-basis motor function. The current mutants, i.e. R397Q, F506C, G575R, A699R, K703Q and K703W are equivalent to R403Q, F513C, G584R, G716R, R719Q and R719W FHC mutants respectively. We measured the molecular-basis force and the sliding velocity generated by these myosin mutants. The measurement revealed that the A699R, K703Q and K703W myosins exhibited the lowest level of force with their preserved actin-activated MgATPase activity. F506C mutant showed the least impairment of the motile and enzymatic activities. The motor function of R397Q and G575R myosins were classified as intermediate. These results suggest that ELC binding domain might be important for force production.

Amino Acid Sequence↗

PubMed1998

Structure-mutation analysis of the ATPase site of Dictyostelium discoideum myosin II.

Three loop structures called the P-loop, switch I loop and switch II loop of myosin are major components of its ATPase site, and share structural and functional homology with the loop structures in other ATPases and GTPases such as kinesin and G-protein. Using the alanine scanning mutagenesis, structure-function relationship of the switch I and switch II loops in Dictyostelium myosin II was examined. Based on crystal structures of Dictyostelium myosin motor domain, functions of each residue in those loops are discussed.

Amino Acid Substitution↗

PubMed1997

Alanine scanning mutagenesis of the switch I region in the ATPase site of Dictyostelium discoideum myosin II.

In order to determine the functional roles of the conserved sequence (NXNSSRFG) of the "switch I" loop (residues 233-240 in Dictyostelium myosin II), alanine scanning mutagenesis was performed on Dictyostelium myosin II. N233A and S237A mutant myosins did not bind a fluorescent analog of ADP, mant-deoxyADP, at the low concentration range (micromolar and had low level of ATPase activities. They were nonmotile when examined by the in vitro motility assay. Dictyostelium cells expressing these myosins showed worse phenotypes than that of myosin-null cells. In contrast to these mutant myosins, R238A myosin tightly bound mant-deoxyADP. However, the mutant had a defect in the ATP hydrolysis step and exhibited the lowest ATPase activities among the mutants examined here. The R238A myosin was nonmotile. R238C or R238H mutations, which mimic the Usher syndrome mutations, generated myosins with similar functional defects to those of the R238A mutation. Cells expressing the R238A myosin exhibited the phenotype similar to that of the myosin-null cells. N235A, S236A, F239A, and G240A myosins retained moderate levels of ATPase activities and could drive sliding of actin filaments at various speeds. Phenotypes of cells expressing them were very similar to that of the wild-type cells. Taken together, these results suggest that side chains of N233 and S237 may play essential roles in holding a nucleotide in the ATPase pocket and that R238 may play crucial roles in the ATP hydrolysis step, while those of the other residues in the switch I loop are not essential for the process.

Adenosine Diphosphate↗

PubMed1997

Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice.

To study the mechanism and risk of human skin cancer from solar light, we exposed human skin transplanted to severe combined immunodeficient mice to daily doses of UVB for periods of approximately 2 years. We have succeeded for the first time in inducing cancer and solar (actinic) keratosis in human skin by UVB. Of 18 normal skins exposed to doses of 7.3 x 10(5) to 1.8 x 10(6) J/m2, 14 actinic keratoses (77.8%) and 3 squamous cell carcinomas (16.7%) developed, whereas neither actinic keratosis nor cancer was observed in 15 human skins not exposed to UVB. Each human skin showed a different susceptibility, and skins sensitive for actinic keratosis were also sensitive for cancer induction. Among p53 mutations at various sites, mutation at codon 242 (C TGC --> C CGC; Cys --> Arg) was specifically observed in both skin cancers and actinic keratoses. Furthermore, double or triple mutations were induced in all UVB-induced skin cancers and in three of eight actinic keratoses. Most of the mutations (17 of 20) occurred at dipyrimidine sites.

Animals↗

PubMed1997

Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II.

The cysteine residue called SH2 in the skeletal myosin heavy chain is conserved among various species. Cys 678 in Dictyostelium myosin II is equivalent to SH2 in skeletal myosin. Using the Dictyostelium myosin II heavy chain gene, SH2 was mutated to Gly, Ala, Ser, or Thr. These mutant myosins were expressed in Dictyostelium myosin-null cells. To investigate how these mutations affect the motor functions of myosin, we examined the phenotypes of the transformed cells. We also purified the mutant myosins, and characterized them by measuring the actin-activated MgATPase activity, sliding velocity of actin filaments and force level. All of these mutant myosins complemented the myosin-specific defects of the myosin-null cells. Consistent with these observed phenotypes, all of the purified mutant myosins retained similar actin-activated MgATPase activities and force levels to those of the wild-type myosin (WT). However, the sliding velocities of actin filaments were significantly different (WT > or = Ser > Ala >> Thr > Gly). In particular, the Gly and Thr mutants exhibited a striking decrease in velocity, while the Ser mutant exhibited velocity comparable to that of the wild-type myosin. Thus, mutations of SH2 resulted in uncoupling of ATP hydrolysis and the sliding.

Actins↗

PubMed1997

Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis.

The gapA gene encoding a novel RasGTPase-activating protein (RasGAP)-related protein was found to be disrupted in a cytokinesis mutant of Dictyostelium that grows as giant and multinucleate cells in a dish culture. The predicted sequence of the GAPA protein showed considerable homology to those of Gap1/Sar1 from fission yeast and the COOH-terminal half of mammalian IQGAPs, the similarity extending beyond the RasGAP-related domain. In suspension culture, gapA- cells showed normal growth in terms of the increase in cell mass, but cytokinesis inefficiently occurred to produce spherical giant cells. Time-lapse recording of the dynamics of cell division in a dish culture revealed that, in the case of gapA- cells, cytokinesis was very frequently reversed at the step in which the midbody connecting the daughter cells should be severed. Earlier steps of cytokinesis in the gapA- cells seemed to be normal, since myosin II was accumulated at the cleavage furrow. Upon starvation, gapA- cells developed and formed fruiting bodies with viable spores, like the wild-type cells. These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis. Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton. Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.

Amino Acid Sequence↗

PubMed1997

Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments.

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by approximately 20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M. ADP.Pi) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP --> M.ADP. Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.

Actin Cytoskeleton↗

PubMed1997

Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.

Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC.

Actins↗

PubMed1997

Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin.

Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, epsilon ADP and TNP-ADP. The complexes of epsilon ADP and BeFx or AIF4- were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.AIF4- and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca(2+)-ATPase (Ki = 1.6 microM). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP approximately ADP.AIF4- approximately ADP.BeFx > ATP gamma S > ADP > PPi. In the case of S1dC, the effects of different nucleotides were smaller and indistinguishable from each other. S1dC bound actin tighter than S1 (Kd = 7 nM and 60 nM, respectively). The actin activated MgATPase activity of S1dC varied between preparations, and the Vmax and K(m) values ranged between 3 and 7 s-1 and 60 and 190 microM, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inactivations at 35 degrees C. These results show that the nucleotide and actin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and the communication between the nucleotide and actin binding sites in these proteins.

Acrylamide↗

PubMed1997

Consecutive maintenance of human solitary and hereditary colorectal polyps in SCID mice.

Recently, the sequential changes from adenoma to adenocarcinoma have been well studied in human colorectal carcinogenesis. To study the precise clonal changes from colorectal polyps to cancer, we have established an experimental system to maintain human colorectal polyps in severe combined immunodeficient (SCID) mice that have been improved by the selective inbreeding of C.B17-scid/scid homozygous male and female showing undetectable serum IgG and IgM (< 1 microgram/ml). Two of two solitary polyps from two nonhereditary colon polyp patients, four of five colon polyps from two Peutz-Jeghers' syndrome patients and one polypoid lesion from a familial polyposis coli (FAP) patient grew very slowly but steadily, at approximately one-tenth the rate of their malignant form, (i.e., adenocarcinoma), in the improved SCID mice and were maintained for a long period (more than 2 years), over several mouse generations. However, two polyps from FAP and Peutz-Jeghers' syndrome patients could not be transplanted further because of microinfection at the transplanted site due to incomplete sterilization of original human tumors prior to surgical operation (endoscopic polypectomy). Transplanted colon polyps had a semitransparent, soft and sticky appearance, with cells containing large amounts of mucin. Malignant transformation of human colon polyp to adenocarcinoma has not been observed during the maintenance period (about 2 years) in SCID mice. In the consecutively maintained human colon polyps, however, K-ras mutations were detected at codon 12, while these mutations were not found in their original polyps in the patients.

Adenomatous Polyposis Coli↗

PubMed1996

Tropomyosin-binding site(s) on the Dictyostelium actin surface as identified by site-directed mutagenesis.

To identify tropomyosin-binding site(s) on the surface of actin molecule, we examined the effect of mutagenesis introduced to subdomain 4 of actin. Because the sequence of Gln228-Ser232 of Dictyostelium actin differs from that of Tetrahymena actin that does not bind tropomyosin, the Dictyostelium/Tetrahymena chimeric actin was produced. Also, Lys238 and Glu241 were replaced with alanine (mutant 645) to study the role of charged residues which are located at both ends of a beta-sheet. As a control experiment, a negative charge was introduced near to the N-terminus (mutant 663). To facilitate the separation of mutant actins without affecting the normal function, Glu360 was replaced with histidine. As a control mutant to such mutants, the mutant 647 (E360H) was produced. Mutant actins were expressed in Dictyostelium cells. All mutant actins were functional: they (i) polymerize and (ii) activate ATPase activity of rabbit skeletal myosin subfragment-1 (S1). The mutant 663 (G2E) showed tropomyosin binding and activated myosin ATPase almost as well as rabbit skeletal actin. However, the tropomyosin binding of the mutant 645 (K238A/E241A/E360H) became magnesium dependent. The chimeric actin (mutant 646: QTAAS-to-KAYKE replacement and E360H) showed decreased tropomyosin binding even in the presence of magnesium ions. These results indicate that the tropomyosin-binding sites of "on"-state actin are on subdomain 4. Surprisingly, the chimeric actin showed more cooperative calcium regulation than rabbit skeletal actin in the presence of tropomyosin-troponin. The mutant actin 645 can hardly activate S1 ATPase irrespective of calcium concentration in the presence of tropomyosin-troponin, even though this actin by itself can activate S1 ATPase. The steric blocking or cooperative/allosteric mechanism of thin filament regulation is discussed.

Actins↗

PubMed1996

Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium.

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d-cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.

Actin Depolymerizing Factors↗

PubMed1995

X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

Adenine↗

PubMed1995

Identification, characterization, and intracellular distribution of cofilin in Dictyostelium discoideum.

We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.

Actin Depolymerizing Factors↗