Biomedical subjects

K Saeki

Publications and source records attributed to K Saeki.

At least 55 records · Page 3Linked to original sources

PubMed2000

Role of residues 230 and 236 of actin in myosin-ATPase activation by actin-tropomyosin.

The Dictyostelium/Tetrahymena-chimeric actin (Q228K/T229A/A230Y) showed higher Ca(2+)-activation of myosin S1 ATPase in the presence of tropomyosin-troponin. The crystal structure of the chimeric actin is almost the same as that of wild-type except the conformation of the side chain of Leu236. Here, we introduced an additional mutation (L236A), in which the side chain of Leu236 was truncated, into the chimeric actin (Q228K/T229A/A230Y/L236A). Without regulatory proteins, the new mutant actin showed normal myosin S1 activation and normal sliding velocity. However, in the presence of tropomyosin, the new mutant actin activated myosin S1 ATPase higher than the wild-type actin and showed higher velocities in in vitro motility assay at low HMM concentrations. These results suggest that the mutations of A230Y and L236A in the actin subdomain-4 facilitate the transition of thin filaments from a "closed" state to an "open" state.

Actins↗

PubMed2000

Analysis of prion protein mRNA by in situ hybridization in brain and placenta of sheep.

In this study, prion protein (PrP) mRNA was focally detected in brain and placenta of pregnant sheep by Northern blot analysis. In addition, host-encoded cellular prion protein (PrP(C)) was observed in brain and placenta of the ruminant by Western blot analysis as well. Localization of PrP mRNA in pregnant sheep tissues was rendered possible with in situ hybridization. In sheep brain, PrP mRNA was predominantly localized within large neocortical neurons in the cerebrum, Purkinje cells and neurons of the molecular and granule cell layers in the cerebellum. In the placenta, signals were observed in the myometrium, including stratum longitudinale tunicae muscles and circular layers of muscular tunics. In the caruncle and placentome, signals were stronger by in situ hybridization. Since accumulation of the scrapie isoform PrP (PrP(Sc)) is required to PrP(C), these results suggest that brain and placenta of sheep may be important organs and sites for the conversion of PrP(C) to PrP(Sc).

Animals↗

PubMed2000

Crystal structure of tobacco necrosis virus at 2.25 A resolution.

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.

Amino Acid Sequence↗

PubMed2000

Histamine is a potent inducer of IL-18 and IFN-gamma in human peripheral blood mononuclear cells.

Histamine (10-7 to 10-4 M) concentration-dependently stimulated the production of IL-18 and IFN-gamma and inhibited the production of IL-2 and IL-10 in human PBMCs. Histamine in the same concentration range did not induce the production of IL-12 at all. The stimulatory or inhibitory effects of histamine on cytokine production were all antagonized by H2 receptor antagonists ranitidine and famotidine in a concentration-dependent manner, but not by H1 and H3 receptor antagonists. Selective H2 receptor agonists, 4-methylhistamine and dimaprit, mimicked the effects of histamine on five kinds of cytokine production. The EC50 values of histamine, 4-methylhistamine, and dimaprit for the production of IL-18 were 1.5, 1.0, and 3.8 microM, respectively. These findings indicated that histamine caused cytokine responses through the stimulation of H2 receptors. All effects of histamine on cytokine responses were also abolished by the presence of either anti-IL-18 Ab or IL-1beta-converting enzyme/caspase-1 inhibitor, indicating that the histamine action is dependent on mature IL-18 secretion and that IL-18 production is located upstream of the cytokine cascade activated by histamine. The addition of recombinant human IL-18 to the culture concentration-dependently stimulated IL-12 and IFN-gamma production and inhibited the IL-2 and IL-10 production. IFN-gamma production induced by IL-18 was inhibited by anti-IL-12 Ab, showing the marked contrast of the effect of histamine. Thus histamine is a very important modulator of Th1 cytokine production in PBMCs and is quite unique in triggering IL-18-initiating cytokine cascade without inducing IL-12 production.

Adjuvants, Immunologic↗

PubMed2000

Citric acid etching of cervical sclerotic dentin lesions: an AFM study.

Atomic force microscopy (AFM) has been used to determine microstructural changes, etching rates of peritubular dentin, and intertubular dentin recession during demineralization in dilute acidic solutions. These studies have not included many forms of altered dentin, including noncarious sclerotic root dentin associated with Cl V (abfraction) lesions. We sought to determine microstructural changes and recession rates during demineralization in citric acid (pH 2.5, 0.018M) for the transparent/sclerotic zone. Highly polished dentin disks were prepared from teeth with noncarious C1 V lesions (n = 3) and compared with normal root dentin (n = 3). Samples were etched at 5-s intervals for 1 min and at longer intervals up to 30 min. The depth changes in various portions of the dentin with respect to the reference layer were measured and changes in microstructure observed in solution in the wet cell of the AFM. In sclerotic dentin, most tubule lumens were occluded with crystalline deposits that etched more slowly than the other dentin components, but etching rates could not be determined due to their irregular geometry. The intertubular dentin recession quickly reached a plateau after a depth change of <1 microm for all dentin types, in agreement with prior work. Mixed linear regression models indicated an important difference between the etching of sclerotic intertubular dentin and that of non-sclerotic root dentin that became apparent after 600 s (p = 0.037). The sclerotic intertubular dentin underwent less depth change at the plateau (558 nm) compared to normal root dentin (744 nm). In addition, normal root dentin underwent significantly greater recession than coronal dentin (p = 0.002). The results of this study indicate that intertubular sclerotic dentin from Cl V lesions etches differently than normal root dentin, and this may help explain the difficulties in restoring such lesions with current bonding procedures.

Acid Etching, Dental↗

PubMed2000

Enhanced expression of cellular prion protein gene by insulin or nerve growth factor in immortalized mouse neuronal precursor cell lines.

In order to understand the fundamental and putative roles of PrP(c) in the central nervous system, neuronal cell lines were established. Cells were immortalized by recombinant retrovirus vector-mediated transduction of SV40 T-antigen gene. Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology. These cell lines showed the properties of neuronal progenitor cells in antigenicity, morphology and responses to differentiating agents. Expression of PrP(c) was detected by immunocytochemical analysis. These cell lines responded to differentiating agents such as dibutyl cyclic AMP (dcAMP) and phorbol 12-myristate 13-acetate (PMA) before developing into neuronal-like cells. Neurite extensions were observed 20 min after incubation with the differentiating agents. Treatment with nerve growth factor (NGF) and insulin induced cell differentiation and enhanced expression of PrP gene (Prnp) mRNA and protein. The latter phenomenon was not inhibited by wortmannin, which is a specific inhibitor of phosphatidylinositol 3-kinase. These results suggest that PrP(c) plays an important role in the differentiation-mediated classic signaling pathway of neuronal cell.

Androstadienes↗

PubMed2000

Structural basis for the higher Ca(2+)-activation of the regulated actin-activated myosin ATPase observed with Dictyostelium/Tetrahymena actin chimeras.

Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca(2+)-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of ATPase activation in the presence of tropomyosin-troponin and Ca(2+) to that without tropomyosin-troponin becomes about four times as large as the ratio for the wild-type actin. To understand the structural basis of this higher Ca(2+)-activation, we have determined the crystal structures of the 1:1 complex of Dictyostelium mutant actins (half chimera-1 and full chimera) with gelsolin segment-1 to 2.0 A and 2.4 A resolution, respectively, together with the structure of wild-type actin as a control. Although there were local changes on the surface of the subdomain 4 and the phenolic side-chain of Tyr230 displaced the side-chain of Leu236 from a non-polar pocket to a more solvent-accessible position, the structures of the actin chimeras showed that the mutations in the 228-232 region did not introduce large changes in the overall actin structure. This suggests that residues near position 230 formed part of the tropomyosin binding site on actin in actively contracting muscle. The higher Ca(2+)-activation observed with A230Y-containing mutants can be understood in terms of a three-state model for thin filament regulation in which, in the presence of both Ca(2+) and myosin heads, the local changes of actin generated by the mutation (especially its phenolic side-chain) facilitate the transition of thin filaments from a "closed" state to an "open" state. Between 394 and 469 water molecules were identified in the different structures and it was found that actin recognizes hydrated forms of the adenine base and the Ca ion in the nucleotide binding site.

Actins↗

PubMed2000

Anti-mutagenic structural modification by fluorine-substitution in highly mutagenic 4-methylquinoline derivatives.

We have previously shown that fluorine-substitution at position 3 of quinoline deprived this molecule of mutagenicity, possibly due to interference with the yield of its metabolically activated form, the 1,4-hydrated 2,3-epoxide (enamine epoxide), which is directly responsible for the mutagenic modification of DNA. To further explore the possibility of a method for anti-mutagenic modification of mutagens by fluorine-substitution, 4-methylquinoline (4-MeQ), the most mutagenic form of all the quinoline derivatives examined so far, was used as a target in the present study. Five mono- and di-fluorinated derivatives of 4-MeQ, 2-fluoro-4-methylquinoline (2-F-4-MeQ), 6-F-4-MeQ, 7-F-4-MeQ, 2,6-difluoro-4-methylquinoline (2, 6-diF-4-MeQ), and 2,7-diF-4-MeQ, were subjected to analysis of their structure-mutagenicity relationships. The 2-fluorinated derivatives (2-F-4-MeQ, 2,6-diF-4-MeQ, and 2,7-diF-4-MeQ) were all non-mutagenic in the Ames test. 7-F-4-MeQ was as highly mutagenic as, and 6-F-4-MeQ was less mutagenic than non-fluorinated 4-MeQ. Metabolic studies were also conducted with 4-MeQ, 2-F-4-MeQ, 6-F-4-MeQ, and 7-F-4-MeQ, using a liver microsomal enzyme fraction prepared from the 3-methylcholanthrene-treated rat. The HPLC analytical data showed that, although the metabolic patterns (hydroxylation at 4-methyl group as a main metabolic pathway and 3-hydroxylation as a minor pathway) of these four F-MeQs were similar to one another, only the 3-hydroxy metabolite of 2-F-4-MeQ was not produced under the present experimental conditions employed. These results suggest that fluorine-substitution at position 2 of 4-MeQ inhibited the formation of the enamine epoxide in the pyridine moiety and deprived this molecule of mutagenicity as in the case of quinoline.

Animals↗

PubMed2000

A230Y mutation of actin on subdomain 4 is sufficient for higher calcium activation of actin-activated myosin adenosinetriphosphatase in the presence of tropomyosin-troponin.

To probe the mechanism by which Ca(2+) activates muscle contraction through tropomyosin and troponin, we have produced mutant actins using Dictyostelium discoideum. We focused on the sequence 228-232 (QTAAS) that is located in subdomain 4 of actin, because the chimera actin in which this sequence was replaced by KAYKE showed not only poorer tropomyosin binding but also the unexpected "higher Ca(2+) activation" [Saeki, K., et al. (1996) Biochemistry 35, 14465-14472]. We found that this higher Ca(2+) activation is solely due to the A230Y mutagenesis. Because A230Y mutant actin showed normal tropomyosin binding, the higher Ca(2+) activation is not the consequence of poorer tropomyosin binding. The significance of these results is discussed in view of a three-state model [McKillop, D. F., Geeves, M. A. (1993) Biophys. J. 65, 693-701].

Actins↗

PubMed2000

Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond.

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.

Animals↗

PubMed2000

Acetylation at the N-terminus of actin strengthens weak interaction between actin and myosin.

The N-terminus of all actins so far studied is acetylated. Although the pathways of acetylation have been well studied, its functional importance has been unclear. A negative charge cluster in the actin N-terminal region is shown to be important for the function of actomyosin. Acetylation at the N-terminus removes a positive charge and increases the amount of net negative charges in the N-terminal region. This may augment the role of the negative charge cluster. To examine this possibility, actin with a nonacetylated N-terminus (nonacetylated actin) was produced. The nonacetylated actin polymerized and depolymerized normally. In actin-activated heavy meromyosin ATPase assays, the nonacetylated actin showed higher K(app) without significantly changing V(max), compared with those of wild-type actin. This is in contrast to the effect of the N-terminal negative charge cluster, which increases V(max) without changing K(app). These results indicate that the acetylation at the N-terminus of actin strengthens weak actomyosin interaction.

Acetylation↗

PubMed2000

Relationship between the concentration of CDDP in tumor and tumor size after isolated lung perfusion treatment experimental study on a solitary pulmonary sarcoma model in rats.

BACKGROUND AND OBJECTIVES: Few investigations on the pharmacokinetics of isolated lung perfusion (ILP) regional chemotherapy in tumors have been conducted. The purpose of this study was to evaluate the pharmacokinetic character of ILP in tumor tissues. Materials and Methods A solitary tumor nodule model was established in Fisher 344 rats by inoculating 1.0 x 10(6) methylcholanthrene-induced sarcoma cells into the left lung. On 14-21 days after the inoculation of tumor cells, rats were randomized into groups subjected to 10, 20, 40 or 60 min ILP with cis-diamminedichloroplatinum (CDDP) at concentrations of 25, 50, or 100 microg/ml. Total platinum concentration in tumors was measured by a flameless atomic absorption spectrophotometer. RESULTS: The average tumor weights and total platinum concentrations were not significantly different among the perfusion groups. We found, however, that the total platinum concentrations in tumor nodules were significantly correlated inversely with the tumor weight (total platinum concentration in tumor = 1.167 x 1/tumor wt; R(2) = 0.981; P < 0.001). CONCLUSIONS: The concentration of total platinum in the tumor tissue was found to be dependent on the weight of the tumor in ILP. Higher concentrations of total platinum were expressed in smaller tumor nodules than larger ones, indicating that ILP may be more effective against small tumors than large ones. Therefore, we suggest that large tumor legions should be debulked before ILP in patients with an unresectable lung tumor.

Animals↗

PubMed2000

Tea catechins and related polyphenols as anti-cancer agents.

Epigallocatechin gallate (EGCg) and theaflavins, a major constituent of green tea infusion and the constituents of black tea, respectively, were found to inhibit matrix metalloproteinases (MMPs) which are intimately associated with tumor invasion and metastasis. EGCg and related polyphenols exhibited apoptosis-inducing activity for several cancer cell lines including human stomach and colon cancer cells. Comparison of the activity of these compounds revealed the importance of the number and the steric disposition of hydroxyl groups. A pyrogallol-type structure in a molecule is a minimum requirement for apoptosis induction of catechin compounds and that in the B ring has an important role in the activity. These data would provide useful information for designing anti-cancer agents on the basis of anti-inhibitory activity for MMPs and/or apoptosis-inducing activity.

Anticarcinogenic Agents↗

PubMed2000

Genomic structure of mouse IA-2: comparison with its human homologue.

AIMS/HYPOTHESIS: IA-2 is a transmembrane protein with a tyrosine phosphatase (PTP)-like structure and a major autoantigen in Type I (insulin-dependent) diabetes mellitus. Because the nucleotide sequence of human and mouse IA-2 cDNA are closely related, it seemed likely that the genomic organization of the two molecules would be similar. To test this possibility the current experiments were initiated to characterize and compare the genomic structure of mouse and human IA-2. METHODS: IA-2 cDNA was used to screen a 129SVJ mouse genomic library. We selected and mapped 7 overlapping clones. The subcloned inserts were used to determine intron-exon junctions by direct sequencing. Polymerase chain reaction and restriction mapping were used to estimate the size of the introns. RESULTS: The mouse IA-2 gene and the 5' upstream regulatory region were isolated and the intron-exon junctions determined. Mouse IA-2 encompasses approximately 20 kb and encodes 23 exons. Both the 3' and 5' ends were mapped by rapid amplification of cDNA ends (RACE) and a 2 kb 5'-upstream region was shown to have functional promoter activity. CONCLUSION/INTERPRETATION: Comparison of the genomic structure of mouse and human IA-2 shows that they have the same number of exons and nearly identical intron-exon junctions. The region around the major transcription start site of mouse IA-2 is similar to human IA-2 and other transmembrane protein tyrosine phosphatases. It is concluded that human and mouse IA-2 are highly conserved and derived from a common ancestral gene.

Amino Acid Sequence↗

PubMed2000

Growth-related changes in heavy metal accumulation in green turtle (Chelonia mydas) from Yaeyama Islands, Okinawa, Japan.

Concentrations of nine heavy metals (Fe, Mn, Zn, Cu, Pb, Ni, Cd, Co, and Hg) were determined in liver, kidney, and muscle of 50 green turtles (Chelonia mydas) collected from Yaeyama Islands, Okinawa, Japan, to elucidate growth-related changes in heavy metal accumulation during different growth stage. Considerably high Cu concentrations were found in the liver of smaller turtles. Mean hepatic concentration of Cu was 50.2 microg/g wet weight which varied widely (4.27-113 microg/g wet weight). Cadmium concentrations decreased with increasing the carapace length. The juvenile green turtles in the pelagic ocean are likely feed on zooplankton, while adult coastal inhabiting green turtles mainly feed on sea grasses and seaweeds. Concentrations of Cd in sea plants are lower than those in zooplankton. The specific accumulation of Cd found in the green turtle seems to be attributable to their feeding habit, which is a shift from carnivore to herbivore at different growth stages.

Aging↗

PubMed2000

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence.

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50 degrees C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl.

Amino Acid Sequence↗

PubMed2000

Importance of a pyrogallol-type structure in catechin compounds for apoptosis-inducing activity.

Several catechin compounds were examined for their ability to induce apoptosis in human histiocytic lymphoma U937 cells. Catechins with a pyrogallol-type structure in a B-ring induced apoptosis and a 3-O-gallate group in cis-relationship to the B ring enhanced the activity. Catechins without a pyrogallol-type structure in a molecule lacked activity. These data suggest the important role of the 5'(3')-hydroxyl group in the B-ring and that a pyrogallol-type structure in a molecule is a minimum requirement for apoptosis induction by catechin compounds.

Apoptosis↗

PubMed2000

Mercury and cadmium in common cormorants (Phalacrocorax carbo).

Mercury (Hg) and cadmium (Cd) in common cormorants (Phalacrocorax carbo) collected in Lake Biwa, Japan and Tokyo, Japan, were investigated to elucidate the biological behaviors of these elements, and to assess exposure to these pollutants of wild, fish-eating birds. Hg and Cd concentrations were highest in the liver and kidneys, respectively. The lowest levels of both elements were observed in chicks. Hg concentrations in all tissues except brain increased significantly with growth from chicks to juveniles (p<0.05, U-test). Cd concentrations in the kidneys and liver also increased significantly during growth from juvenile to adult (p<0.005, U-test). When comparing hepatic Hg and Cd in adult birds between 10 samples from Lake Biwa and nine samples from Tokyo, the Cd concentrations in the Lake Biwa samples (1.4+/-0.37 microg/g dry wt) (mean+/-SD) were significantly higher than those from Tokyo (0.32+/-0.16 microg/g dry wt) (p<0.005, U-test), while no statistically significant difference was found in the Hg concentrations. Possible causes of these differences were discussed in relation to their prey.

Journal Article↗