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K Yamane

Publications and source records attributed to K Yamane.

At least 127 records · Page 7Linked to original sources
PubMed1997

A DNA-topoisomerase-II-binding protein with eight repeating regions similar to DNA-repair enzymes and to a cell-cycle regulator.

A two-hybrid system was used to isolate factors that interact with the C-terminal region of DNA topoisomerase IIbeta. A positive clone isolated from a HeLa cDNA library encoded 1522 amino acid residues (molecular mass 170670). The protein, designated topoisomerase-IIbeta-binding protein 1 (TopBP1), interacted with the C-terminal region of topoisomerase IIbeta synthesized in vitro. A database search indicated that TopBP1 possessed eight regions similar to regions of Rad4, Cut5, Ect2, Rev1 and X-ray repair cross-complementing 1 (XRCC1) proteins and a region similar to auto-modification sites of poly(ADP-ribose) polymerase, suggesting that TopBP1 supported catalytic reactions of topoisomerase II through transient breakages of DNA strands.

Amino Acid Sequence↗
PubMed1997

Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities. Constitutive and temporally controlled expression of different sip genes.

Most biological membranes contain one or two type I signal peptidases for the removal of signal peptides from secretory precursor proteins. In this respect, the Gram-positive bacterium Bacillus subtilis seems to be exceptional, because it contains at least four chromosomally-encoded type I signal peptidases, denoted SipS, SipT, SipU, and SipV. Here, we report the identification of the sipT and sipV genes, and the functional characterization of SipT, SipU, and SipV. The four signal peptidases have similar substrate specificities, as they can all process the same beta-lactamase precursor. Nevertheless, they seem to prefer different pre-proteins, as indicated by studies on the processing of the pre-alpha-amylase of Bacillus amyloliquefaciens in strains lacking SipS, SipT, SipU, or SipV. The sipU and sipV genes are constitutively transcribed at a low level, suggesting that they are required for processing of (pre-)proteins secreted during all growth phases. In contrast, the transcription of sipS and sipT is temporally controlled, in concert with the expression of the genes for most secretory proteins, which suggests that SipS and SipT serve to increase the secretory capacity of B. subtilis. Taken together, our findings suggest that SipS, SipT, SipU, and SipV serve different functions during the exponential and post-exponential growth phase of B. subtilis.

Amino Acid Sequence↗
PubMed1997

Identification of a region required for binding to presecretory protein in Bacillus subtilis Ffh, a homologue of the 54-kDa subunit of mammalian signal recognition particle.

Bacillus subtilis Ffh protein is a homologue of the 54-kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C-terminal methionine-rich domain (M-domain). Two of the hydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma, S., Takamatsu, H., Nakamura, K., & Yamane, K. (1996) J. Biol. Chem. 271, 13,140-13,146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311-Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121-Lys279, Lys364-Met446, or Leu338-Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh proteins, FfhQQQR and FfhQQQQ having wild-type residues 398-401 (Arg-Arg-Lys-Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQQR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQQR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including h1 is required for B. subtilis Ffh to bind to presecretory protein. The results also suggest that scRNA is required for the complete function of the B. subtilis SRP-like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA.

Amino Acid Sequence↗
PubMed1997

High fat, high cholesterol diets alter low density lipoprotein size and binding affinity in monkeys.

The purpose of this study was to examine the effects of various dietary fats on low density lipoprotein (LDL) binding in an in vitro system where receptor number is not regulated. Cynomolgus monkeys were fed diets containing 37% of energy from fat, with various degrees of saturation, and 0.4 mg/kcal cholesterol or low-fat (13% of energy), low cholesterol (0.03 mg/kcal) chow. Plasma LDL was isolated after 16 weeks. The fatty acid composition of LDL showed enrichment corresponding to the dietary fats consumed, and the high fat, high cholesterol diets produced marked hypercholesterolemia compared to chow feeding. Of those fed the high fat diets, monkeys fed the fish oil diet had the highest LDL cholesterol concentrations, 13.25 +/- 0.77 mmol/l, while those fed the safflower oil diet had the lowest, 7.51 +/- 3.31. LDL from chow fed monkeys had the lowest binding affinity; the Kd was 26.2 +/- 8.7 microg/ml, nearly twice that of the high fat diets (P = 0.003). No significant differences in binding were found between the different high fat diets, although there was a trend toward lower affinity in the diets enriched in polyunsaturated fat. LDL size was affected by diet with chow fed monkeys having the smallest average LDL, 259.3 +/- 1.7 A compared to the other groups (P = 0.03). Monkeys fed the fish oil diet tended to have smaller LDL, but this was not significantly different from the other high fat diets. Binding affinity was correlated with LDL size, r = 0.54, P < 0.01. LDL composition, as measured by apo B/cholesterol ratio, was altered by feeding a high fat, high cholesterol diet. The ratio was reduced in the LDL samples from monkeys fed the high fat diets compared to those fed chow, but this ratio was not significantly correlated with binding. Thus, it appears that increasing dietary fat and cholesterol intake increases LDL size and binding affinity, such that LDL metabolism may be altered independently from effects on receptor number; the type of dietary fat does not seem to influence this process when fat and cholesterol content is very high.

Animals↗
PubMed1997

Optical isometric force transducer for measurement of rat skeletal muscle contraction in the NMR spectrometer.

In order to measure force of contraction of the rat gastrocnemius muscle in the NMR spectrometer with a simultaneous observation of 31P NMR spectra, an optical force transducer was constructed using gratings and optical fibers. Muscle contraction was accurately recorded in the spectrometer without interfering with NMR measurements. The half height width of the phosphocreatine (PCr) peak in the presence or absence of the transducer was 5 Hz. Decrease in PCr and increase in inorganic phosphate (Pi) in the muscle were successfully followed during monitoring of the force developed during exercise. The change in the PCr:(PCr + Pi) ratio correlated well with the time-force integral from the optical force transducer. In general, there was no interference with the 31P NMR spectrum when the transducer was attached to rat leg, so this optical force transducer should prove valuable for studying muscle physiology in the NMR spectrometer.

Animals↗
PubMed1997

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice.

CD8+ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2Db restricted manner. Although these results suggest that CD8+ T cells recognize peptides derived from DQ6 molecule bound to H-2Db on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6 alpha or beta chain and carrying the motifs of Db-binding peptides, and examined their capacity to induce cytotoxicity in the CD8+ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8+ T cells when this peptide was pulsed to H-2Db expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8+ T cells in the context of H-2Db molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4+ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8+ T cells.

Animals↗
PubMed1997

Differential requirement of MHC class II molecules expressed on hematopoietic cells for positive selection of CD4+ thymocytes in TCR alpha beta and TCR beta transgenic mice.

To address whether expression of the relevant MHC molecules on thymic epithelial cells (TEC) is sufficient for positive selection, the fate of CD4+ T cells directed by the DR alpha E beta b molecule was compared between two lines of HLA-DRA transgenic mice, DR alpha-24 expressing DR alpha E beta b on TEC and hematopoietic cells, and DR alpha-30 expressing the molecule on TEC but not on other cell lineages. CD4+ T cells were selected to mature on DR alpha E beta b molecules in both lines deficient for the endogenous MHC class I and class II molecules, whereas CD4+ T cell differentiation in DR alpha-30, as compared with that in DR alpha-24, was severely impaired when these mice were crossed with mice transgenic for the DR alpha E beta b-restricted alpha beta TCR. In contrast, CD4+ T cells expressing the same TCR alpha beta as the transgenes were selected to mature in DR alpha-30 crossed with the single TCR beta transgenic mice. Taken together, these results indicate that the restricted expression of DR alpha E beta b on TEC is sufficient for positive selection in the TCR beta and non-TCR transgenic mice, but the expression on both TEC and hematopoietic cells is required for that in the TCR alpha beta transgenic mice. In the light of the difference in the developmental stage at which alpha beta TCR are expressed between TCR alpha beta and TCR beta transgenic mice, the interaction of the TCR alpha beta with DR alpha E beta b molecules expressed on hematopoietic cells required for positive selection in TCR alpha beta transgenic mice could be mapped to the early stage of T cell differentiation and might mimic 'undefined' early signaling of thymocytes in physiological T cell differentiation.

Animals↗
PubMed1997

[Angiographical evaluation of extracranial carotid artery: comparison between Japanese and Hungarian].

We investigated the cervical level of carotid bifurcation, diameter of common carotid artery (CCA) and internal carotid artery (ICA) in 147 Japanese patients (150 arteries, range of age 31-79) and in 490 Hungarian patients (517 arteries, range of age 12-77). The cervical level of carotid bifurcation was determined by comparison with cervical vertebra. The carotid bifurcation of Japanese was most frequent at the lower part of 3rd cervical vertebra but in Hungarian the bifurcation was most frequent at the middle part of 4th cervical vertebra. Japanese carotid bifurcation was high-positioned compared with Hungarian. The mean CCA diameter in Japanese was 7.47 mm in male and 7.07 mm in female, in Hungarian 9.24 mm in male and 7.80 mm in female. The mean ICA diameter in Japanese was 4.96 mm in male and 4.83 mm in female, in Hungarian 8.56 mm in male and 7.66 mm in female. CCA and ICA diameter were larger in male than in female. The diameter of CCA had a significantly positive correlation with age (p < 0.002) but ICA had no similar correlation.

Adolescent↗
PubMed1996

X-ray structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011. Comparison of two independent molecules at 1.8 A resolution.

Cyclodextrin glucanotransferase (CGTase) is an enzyme which produces cyclodextrins by the degradation of starch. The enzyme from alkalophilic Bacillus sp. 1011, consisting of 686 amino acid residues, was crystallized from the solution containing 20% PEG 3000 and 20% 2-propanol at pH 5.6 adjusted with citrate buffer. The space group was P1 and the unit cell contained two molecules (V(m) = 2.41 A(3) Da(-1)). The structure was solved by the molecular replacement method and refined to a conventional R value of 0.161 (R(free) = 0.211) for the reflections in the resolution range 1.8-10 A by energy minimization combined with simulated annealing. The molecule consists of five domains, designated A-E, and its backbone structure is similar to the structure of other bacterial CGTases. The molecule has two calcium binding sites where calcium ions are coordinated by seven ligands, forming a distorted pentagonal bipyramid. The two independent molecules are related by a pseudotwofold symmetry and are superimposed with an r.m.s. deviation value of 0.32 A for equivalent C(alpha) atoms. Comparison of these molecules indicated the relatively large mobility of domains C and E with respect to domain A. The active site is filled with water molecules forming a hydrogen-bond network with polar side-chain groups. Two water molecules commonly found in the active center of both molecules link to several catalytically important residues by hydrogen bonds and participate in maintaining a similar orientation of side chains in the two independent molecules.

Journal Article↗
PubMed1996

Bacillus subtilis Ffh, a homologue of mammalian SRP54, can intrinsically bind to the precursors of secretory proteins.

We analyzed the binding activity of B. subtilis Ffh to the precursors of secretory proteins by purifying mature and precursor proteins of beta-lactamase derived from pUC18 and its derivatives, of which the signal peptide region was replaced with that of E. coli OmpA, B. subtilis AprE, PBP5* or an alkalophilic Bacillus sp. #1011 CGTase. Each of them was mixed with purified B. subtilis Ffh in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The tested precursor proteins, including those of E. coli, of which the signal sequences differ from those of B. subtilis in the number of charged amino acids and hydrophobicity, cross-linked with Ffh, whereas mature proteins did not. The addition of scRNA, the B. subtilis counterpart of mammalian SRP 7S RNA, into the mixture did not affect the complex formation. These findings suggest that B. subtilis Ffh intrinsically binds to several precursor proteins.

Amino Acid Sequence↗
PubMed1996

The effect of Srb, a homologue of the mammalian SRP receptor alpha-subunit, on Bacillus subtilis growth and protein translocation.

To determine the signal recognition particle (SRP)-SRP receptor (Srb) system in Bacillus subtilis (Bs), we cloned the Bs srb gene, which encodes a homologue of the mammalian SRP receptor alpha-subunit [Oguro et al., DNA Res. 2 (1995) 95-100]. We sequenced a 6098-bp DNA containing srb and analyzed the gene organization. Primer extension experiment and Northern blot analysis revealed that srb constitutes an operon with two additional ORFs. A database search of known proteins revealed that one encodes a homologue of Escherichia coli RNase III [36.0% identical amino acids (aa)] and the other encodes a homologue of yeast Smc1 (26.6% identical aa). We then constructed a Bs mutant in which srb expression was induced by IPTG. The depletion of Srb caused a defect in the cell growth and the cells became filamentous and twisted. Furthermore, pulse-chase experiments using this mutant revealed that the 17% of the beta-lactamase precursor accumulated in the cell after a 4-min chase in the absence of IPTG, although almost all of the precursors were converted into the mature from after a 1-min chase in the presence of IPTG.

Amino Acid Sequence↗
PubMed1996

Identification of a region of Bacillus subtilis Ffh, a homologue of mammalian SRP54 protein, that is essential for binding to small cytoplasmic RNA.

Bacillus subtilis Ffh and scRNA are homologues of mammalian SRP54 and SRP RNA, respectively, which are components of the eukaryotic signal recognition particle (SRP). Ffh (446 amino acids) interacts with scRNA to form a stable complex in vivo. Here, we identified an RNA-binding domain of Ffh. The results obtained using a series of deletion mutants show that amino acid positions 364 to 432 in the C-terminal region of Ffh correlates with its ability to bind RNA. The amino acid sequence of this region is well conserved among members of the SRP54 protein family. This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407. Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not. The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh. The two glycine residues of the 405GSG407 were also essential. MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not. The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.

Amino Acid Sequence↗
PubMed1996

Identification of protein synthesis elongation factor G as a 4.5 S RNA-binding protein in Escherichia coli.

Escherichia coli 4.5 S RNA is metabolically stable and abundant. It consists of 114 nucleotides, and it is structurally homologous to domain IV of mammalian signal recognition particle (SRP) RNA. In this study, we found two 4.5 S RNA-binding proteins in cell extracts by means of a gel mobility shift assay. One protein was identified as Ffh, which has been characterized as 4.5 S RNA-binding protein. The other protein was separated from Ffh by two consecutive column chromatographic elutions and by monitoring the 4.5 S RNA binding activity. After the second chromatography, a dominant protein with an approximate molecular weight of 78,000 was associated with 4.5 S RNA binding activity. A sequence of the NH2-terminal 19 residues of the 78-kDa protein was completely identical to that of the protein elongation factor G (EF-G) of E. coli, and further it cross-reacted with antiserum against E. coli EF-G. The results obtained using a synthetic oligo RNA corresponding to the 23 S rRNA defining the EF-G binding site indicated that 4.5 S RNA and 23 S rRNA are competitive in 4.5 S RNA binding and that a decanucleotide sequence conserved between them serves as a binding site for EF-G. Conservation of the SRP RNA binding activity of EF-G from Bacillus subtilis suggests that the binding of EF-G to SRP RNA is essential for its function.

Amino Acid Sequence↗
PubMed1996

Fatty acid modification of membrane fluidity in Chinese hamster ovary (TR715-19) cells.

Dietary saturated fatty acids, especially lauric (12:0), myristic (14:0) and palmitic (16:0) acids, which are hypercholesterolemic, influence cell membrane fatty acid composition and affect LDL receptor function. When membrane phospholipid fatty acids in Chinese hamster ovary cells, containing the human LDL receptor, were modified (Hannah J. S. et al., 1995 Metabolism 44, 1428-1434), LDL receptor function was affected, but correlations with DPH-determined membrane fluidity were weak. The role of fluidity in various membrane domains with respect to the LDL receptor is examined here. Membrane fluidity was assessed by measuring steady-state fluorescence polarization of diphenylhexatriene (DPH) and its polar propionic acid (DPH-PA) and trimethylammonium (TMA-DPH) derivatives from 38 to 4 degrees C in fatty acid modified Chinese hamster ovary cells. Fatty acid changes modulated mid-bilayer fluidity as determined with DPH, but fluidity in phospholipid headgroup domains, assessed with DPH-PA and TMA-DPH, was independent of fatty acyl composition. The DPH fluidity was related to membrane unsaturation (P < 0.02), oleate contents (P < 0.009) in particular, but inversely related (P < 0.0002) to the longer chain (> or = 20 C atoms) unsaturated fatty acids with from four to six double bonds. The LDL binding was independent of fluidity, but there were weak relations between LDL internalization and DPH-PA anisotropy and between LDL degradation and TMA-DPH anisotropy. It was concluded that LDL binding was not related to mid-bilayer fluidity, but the results with the polar probes suggest a role of fluidity in modulating vertical displacement of the LDL/LDL receptor complex across the plasma membrane.

Animals↗
PubMed1996

Effect of the expression of DR alpha E beta NOD molecule on the development of insulitis and diabetes in the non-obese diabetic (NOD) mouse.

Previous studies have shown that a transgenic I-E alpha gene, the mouse homologue of human DR alpha gene, prevents the development of insulitis and hence of diabetes in NOD mice. To investigate the mechanism of this prevention, we generated two strains of NOD mice expressing DR alpha E beta molecule: DR alpha-24-NOD expressing DR alpha E beta molecule on thymic epithelial cells (TEC) and bone marrow-derived cells (BDC), and DR alpha-30-NOD expressing DR alpha E beta molecule only on the TEC, and these mice were monitored for disease development. Because the DR alpha E beta molecule reconstituted I-E controlled immune regulation, it would become clear which cell type, TEC or BDC, was responsible for the I-E-mediated disease protection. To our surprise, however, DR alpha-24-NOD developed insulitis and diabetes comparably to non-transgenic littermates. This suggested that the difference in structure between DR alpha and E alpha molecules contributed to the difference in preventive effect on the development of insulitis and diabetes between DR alpha-24-NOD and E alpha-NOD. In an analysis of the T cell proliferative responses to glutamic acid decarboxylase (GAD) 65-derived peptides which were known to be diabetogenic autoantigens, it was shown that DR alpha-24-NOD and NOD acquired comparable level of T cell response to GAD 509-528 but 5-10-fold higher response was observed in E alpha-NOD. This suggested that I-ANOD and E alpha E beta NOD molecules could present GAD 509-528 peptide to T cells, while DR alpha E beta NOD could not. Furthermore, T cells from DR alpha transgenic mice showed proliferative response to antigen-presenting cells from E alpha transgenic mice in primary mixed lymphocyte reaction. This also suggested that the E alpha E beta molecule does differ in structure and peptide binding from the DR alpha E beta molecule. Present data suggested a possibility that the T cell repertoire selection, or the T cell response to GAD 65 and/or other unknown antigens specifically mediated by I-E molecule, may contribute to the prevention of disease development in E alpha-NOD.

Amino Acid Sequence↗