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Biomedical subjects

K Akasaka

Publications and source records attributed to K Akasaka.

At least 19 recordsLinked to original sources
PubMed2004

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure.

Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken beta-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken beta-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.

Acetylation↗
PubMed2004

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.

Adaptation, Physiological↗
PubMed2001

Native-like tertiary structure formation in the alpha-domain of a hen lysozyme two-disulfide variant.

Structure formation in two species of the two-disulfide variant of hen lysozyme was investigated by means of CD spectroscopy, disulfide exchange measurement, and 1H-NMR spectroscopy. One species, 2SS [6-127, 30-115], which contained the two disulfide bonds found in the alpha-domain of authentic lysozyme, had amounts of secondary and tertiary structures, and bacteriolytic activity comparable to those of authentic lysozyme, and showed a cooperative thermal unfolding. By contrast, the other species, 2SS [64-80, 76-94], which contained the beta-domain disulfide bond as well as the inter-domain one, had a limited amount of secondary structure and little tertiary structure. Disulfide-exchange did not occur for 2SS [6-127, 30-115], whereas it occurred for 2SS [64-80, 76-94], indicating that the protein main-chain fold coupled with the formation of two disulfide bonds is relatively stable for the former variant, while unstable for the latter. 1H-NMR spectra of 2SS [6-127, 30-115] showed that native-like local environment is present within the region that corresponds to the alpha-domain, while it is absent within the region that corresponds to the beta or inter-domain. These results indicate that the alpha-domain of hen lysozyme can be an independent folding domain at equilibrium. Although the bipartite nature in the structure formation of hen lysozyme is similar to that reported for alpha-lactalbumin, differences exist between the disulfide-intermediates of the two proteins in terms of the structural domain that accomplishes tertiary structure.

Animals↗
PubMed2001

Two folded conformers of ubiquitin revealed by high-pressure NMR.

High-pressure 15N/1H two-dimensional NMR spectroscopy has been utilized to study conformational fluctuation of a 76-residue protein ubiquitin at pH 4.5 at 20 degrees C. The on-line variable pressure cell technique is used in conjunction with a high-field NMR spectrometer operating at 750 MHz for 1H in the pressure range between 30 and 3500 bar. Large, continuous and reversible pressure-induced 1H and 15N chemical shifts were observed for 68 backbone amide groups, including the 7.52 ppm 15N shift of Val70 at 3500 bar, indicating a large-scale conformational change of ubiquitin with pressure. On the basis of the analysis of sigmoid-shaped pressure shifts, we conclude that ubiquitin exists as an equilibrium mixture of two major folded conformers mutually converting at a rate exceeding approximately 10(4) s(-1) at 20 degrees C at 2000 bar. The second conformer exists at a population of approximately 15% (DeltaG(0) = 4.2 kJ/mol) and is characterized with a significantly smaller partial molar volume (DeltaV(0) = -24 mL/mol) than that of the well-known basic native conformer. The analysis of 1H and 15N pressure shifts of individual amide groups indicates that the second conformer has a loosened core structure with weakened hydrogen bonds in the five-stranded beta-sheet. Furthermore, hydrogen bonds of residues 67-72 belonging to beta5 are substantially weakened or partially broken, giving increased freedom of motion for the C-terminal segment. The latter is confirmed by the significant decrease in 15N[1H] nuclear Overhauser effect for residues beyond 70 at high pressure. Since the C-terminal carboxyl group constitutes the reactive site for producing a multi-ubiquitin structure, the finding of the second folded conformer with a substantially altered conformation and mobility in the C-terminal region will shed new light on the reaction mechanism of ubiquitin.

Magnetic Resonance Spectroscopy↗
PubMed2001

High-pressure NMR study of the complex of a GTPase Rap1A with its effector RalGDS. A conformational switch in RalGDS revealed from non-linear pressure shifts.

Unusually large non-linear 1H and 15N nuclear magnetic resonance chemical shifts against pressure have been detected for individual amide groups of the Ras-binding domain of Ral guanine dissociation stimulator (GDS). The non-linear response is largest in the region of the protein remote from the Rap1A-binding site, which increases by about two-fold by the complex formation with its effector protein Rap1A. The unusual non-linearity is explained by the increasing population of another conformer (N'), lying energetically above the basic native conformer (N), at higher pressure. It is considered likely that the conformational change from N to N' in the Ras-binding domain of RalGDS works as a switch to transmit the effector signal further to molecules of different RalGDS-dependent signaling pathways.

Models, Molecular↗
PubMed2001

Filling a cavity dramatically increases pressure stability of the c-Myb R2 subdomain.

Cavities or packing defects in proteins may generally be related with the dynamics and function of a protein. In the c-Myb R2 subdomain, its single cavity has been shown to be crucial for its DNA recognition. Cavities are also considered important in determining the pressure stability of a protein. In the present work, high-pressure proton nuclear magnetic resonance ((1)H NMR) spectroscopy at 750 MHz is used to study the effect of a cavity-filling mutation (V103L) on the stability of the c-Myb R2 subdomain in the pressure range between 1 and 3,700 bar at 5 degrees C. A dramatic increase in the pressure stability of the c-Myb R2 subdomain is attained, from which we estimate the cavity size to be 35.3 A(3), in good agreement with literature values. We also evaluated the increase in thermodynamic stability DeltaG(0)(1bar) from 5.35 kJ/mol to 7.34 kJ/mol by the mutation, giving a clear example of the effect of a cavity on the global stability of a globular protein.

Magnetic Resonance Spectroscopy↗
PubMed2001

1H NMR application for characterizing water-soluble organic compounds in urban atmospheric particles.

Water-soluble organic compounds (WSOC) in urban atmospheric particles separated by particle size were analyzed by 1H NMR. This is the first utilization of 1H NMR for characterizing WSOC in atmospheric particles. The WSOC dissolved in D2O were analyzed without a separation procedure. Twelve low molecular weight WSOC could be identified and their atmospheric concentrations determined. One of these, monomethyl hydrogen sulfate (MHS), was first detected in an urban area where no oil or coal power plant existed. Methanesulfonic acid (MSA) and hydroxymethanesulfonic acid (HMSA) were detected as major organosulfur compounds. Relatively high concentrations of these low molecular weight WSOC were observed in the particle diameter range of 0.43-1.1 microns. Many complex signals at 3-4 ppm in the NMR spectrum were seen only for the coarse particle samples (1.1 microns < particle diameter). Mannitol was believed to exist in the coarse particles as a major polyol corresponding to these signals. On the other hand, a large broad signal, observed at 2.5-3 ppm, was mostly present in the fine particles. Finally, it was believed that a major part of the WSOC in urban atmospheric fine particles is attributed to ketocarboxylic acids, ketodicarboxylic acids, and dicarboxylic acids.

Air Pollutants↗
PubMed2001

TAG-1-deficient mice have marked elevation of adenosine A1 receptors in the hippocampus.

TAG-1 is a neural recognition molecule in the immunoglobulin superfamily that is predominantly expressed in the developing brain. Several lines of evidence suggest that TAG-1 is involved in the outgrowth, guidance, and fasciculation of neurites. To directly assess the function of TAG-1 in vivo, we have generated mice with a deletion in the gene encoding TAG-1 using homologous recombination in embryonic stem cells. Gross morphological analysis of the cerebellum, the spinal cord, and the hippocampus appeared normal in TAG-1-deficient mice. However, TAG-1 (-/-) mice showed the upregulation of the adenosine A1 receptors determined by [(3)H]cyclopentyl-1,3-dipropylxanthine in the hippocampus, and their greater sensitivity to convulsant stimuli than that in TAG-1 (+/+) mice. We suspect that the subtle changes in neural plasticity induced by TAG-1 deficiency during development cause the selective vulnerability of specific brain regions and the epileptogenicity in TAG-1 (-/-) mice.

Animals↗
PubMed2001

High pressure NMR reveals a variety of fluctuating conformers in beta-lactoglobulin.

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta-lactoglobulin at pH 2.0 and 36 degrees C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N') of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta-barrel, i.e. the hydrophobic core side (betaF-H) (producing I2) and the non-core side (betaB-E) (producing I1). At 1 bar the stability is higher for the core region (DeltaG0 = 6.5(+/-2.0) kcal/mol) than for the non-core region (4.6(+/-1.3) kcal/mol), but at high pressure the stability is reversed due to a larger DeltaV value of unfolding for the core region (90.0(+/-35.2) ml/mol) than that for the non-core region (57.4(+/-14.4) ml/mol), possibly due to an uneven distribution of cavities. The DeltaG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of beta-lactoglobulin, notably N, N', I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.

Animals↗
PubMed2001

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Animals↗
PubMed2001

Pressure-dependent changes in the structure of the melittin alpha-helix determined by NMR.

A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small alpha-helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H-O distance of the NH-O=C hydrogen bonds decreased by 0.021 +/- 0.039 A, leading to an overall compression along the entire helix of about 0.4 A, corresponding to a static compressibility of 6 x 10(-6) bar(-1). The backbone dihedral angles phi and psi are altered by no more than +/- 3 degrees for most residues with a negative correlation coefficient of -0.85 between phi(i) and psi(i - 1), indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies.

Amides↗
PubMed2001

Brachyury homolog (HpTa) is involved in the formation of archenteron and secondary mesenchyme cell differentiation in the sea urchin embryo.

Sea urchin Brachyury homolog (HpTa) is expressed exclusively in the vegetal plate and secondary mesenchyme cells in the embryos of sea urchin Hemicentrotus pulcherrimus. In order to gain insights into the role of HpTa during sea urchin development, we designed experiments to perturb the embryo by inducing ectopic overexpression of HpTa by injecting fertilized eggs with HpTa mRNA. The overexpression of HpTa resulted in suppression of the formation of vegetal plate and secondary mesenchyme cells. We assume that the interaction of HpTa with unknown factors is required for the activation of the HpTa target genes, and that the excess amount of HpTa proteins produced from injected HpTa mRNA depletes the co-factors. In consequence, the target genes of HpTa would be repressed by the overexpression of HpTa. We suggest that HpTa is involved in the formation of the vegetal plate and the differentiation of secondary mesenchyme cells.

Journal Article↗
PubMed2001

Response of native and denatured hen lysozyme to high pressure studied by (15)N/(1)H NMR spectroscopy.

High-pressure (15)N/(1)H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 M urea, over the pressure range 30--2000 bar. Most (1)H and (15)N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 +/- 0.101 p.p.m. ((1)H) and 0.51 +/- 0.36 p.p.m. ((15)N). The shifts indicate that the hydrogen bonds formed to carbonyl groups or water molecules by the backbone amides are, on average, shortened by approximately 0.02 A as a result of pressure. In native lysozyme, six residues in the beta domain or at the alpha/beta domain interface have anomalously large nonlinear (15)N and (1)H chemical-shift changes. All these residues lie close to water-containing cavities, suggesting that there are conformational changes involving these cavities, or the water molecules within them, at high pressure. The pressure-induced (1)H and (15)N shifts for lysozyme denatured in 8 M urea are much more uniform than those for native lysozyme, with average backbone amide shifts of 0.081 +/- 0.029 p.p.m. ((1)H) and 0.57 +/- 0.14 p.p.m. ((15)N). The results show that overall there are no significant variations in the local conformational properties of denatured lysozyme with pressure, although larger shifts in the vicinity of a persistent hydrophobic cluster indicate that interactions in this part of the sequence may rearrange. NMR diffusion measurements demonstrate that the effective hydrodynamic radius of denatured lysozyme, and hence the global properties of the denatured ensemble, do not change detectably at high pressure.

Animals↗
PubMed2000

High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase.

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.

Binding Sites↗
PubMed2000

Development of phosphine reagents for the high-performance liquid chromatographic-fluorometric determination of lipid hydroperoxides.

Phosphine reagents were designed and synthesized as a new type of fluorescent reagents for the determination of lipid hydroperoxides in foodstuff and biological materials. All phosphine reagents prepared had no fluorescence but their oxides, which were produced by the reaction of the phosphines with hydroperoxides, had strong fluorescence. Among the phosphine reagents prepared, diphenyl-1-pyrenylphosphine had the most suitable properties as a fluorescent reagent and was successfully applied to the determination of hydroperoxides by batch, flow injection and HPLC post-column methods.

Chromatography, High Pressure Liquid↗