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Biomedical subjects

K Akasaka

Publications and source records attributed to K Akasaka.

At least 55 records · Page 3Linked to original sources
PubMed1999

Effect of pressure on the tertiary structure and dynamics of folded basic pancreatic trypsin inhibitor.

The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein.

Aprotinin↗
PubMed1999

HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo.

The mechanism of micromere specification is one of the central issues in sea urchin development. In this study we have identified a sea urchin homologue of ets 1 + 2. HpEts, which is maternally expressed ubiquitously during the cleavage stage and which expression becomes restricted to the skeletogenic primary mesenchyme cells (PMC) after the hatching blastula stage. The overexpression of HpEts by mRNA injection into fertilized eggs alters the cell fate of non-PMC to migratory PMC. HpEts induces the expression of a PMC-specific spicule matrix protein, SM50, but suppresses of aboral ectoderm-specific arylsulfatase and endoderm-specific HpEndo16. The overexpression of dominant negative delta HpEts which lacks the N terminal domain, in contrast, specifically represses SM50 expression and development of the spicule. In the upstream region of the SM50 gene there exists an ets binding site that functions as a positive cis-regulatory element. The results suggest that HpEts plays a key role in the differentiation of PMCs in sea urchin embryogenesis.

Amino Acid Sequence↗
PubMed1999

Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos.

In spite of their potential importance in evolution, there is little information about Hox genes in animal groups that are related to ancestors of deuterostome. It has been reported that only two Hox genes (Hbox1 and Hbox7) are expressed significantly in sea urchin embryos. Expression of Hbox1 protein is restricted to the aboral ectoderm, and Hbox7 expression is restricted to oral ectoderm, endoderm and secondary mesenchyme cells in sea urchin embryos after the gastrula stage. With the aim of gaining insight into the role of Hbox1 and Hbox7 in sea urchin development, Hbox1 and Hbox7 overexpression experiments were performed. Overexpression of Hbox1 repressed the development of oral ectoderm, endoderm and mesenchyme cells. On the contrary, overexpression of Hbox7 repressed the development of aboral ectoderm and primary mesenchyme cells. The data suggest that Hbox1 and Hbox7 are expressed in distinct non-overlapping territories, and overexpression of either one inhibits territory-specific gene expression in the domain of the other. It is proposed that an important function of both Hbox1 and Hbox7 genes is to maintain specific territorial gene expression by each one, in its domain of expression, while repressing the expression of the other in this same domain.

Amino Acid Sequence↗
PubMed1999

Lim1 related homeobox gene (HpLim1) expressed in sea urchin embryos.

A cDNA clone for the LIM class homeobox gene (HpLim1) of the sea urchin, Hemicentrotus pulcherrimus, was isolated. HpLim1 contains two LIM domains and a LIM-class homeodomain, and amino acid sequences of these three domains are highly homologous to corresponding domains of Lim1 of other animals. Accumulation of HpLim1 transcripts begins at hatching, and declines after the mesenchyme blastula stage. HpLim1 mRNA was localized in the vegetal plates of hatched blastulae, but it was not detectable in primary mesenchyme cells (PMC) ingressed into the blastocele. HpLim1 mRNA-injected embryos became spherical with markedly reduced gut formation, failed to express marker proteins for aboral ectoderm and mesoderm, and mainly expressed an oral ectoderm marker. These results imply that while short-term expression of HpLim1 in the vegetal plate is needed for differentiation of aboral ectoderm, endoderm and PMC, ectopic expression of HpLim1 suppresses normal differentiation directing all embryonic cells to differentiate into oral ectoderm.

Amino Acid Sequence↗
PubMed1999

Secondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitor.

Based on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C(alpha) signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta3 strand is completely missing and the alpha2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative. Higher protection against hydrogen exchange for residues in part of the beta4 strand implies that this region might serve as a folding core.

Amino Acid Sequence↗
PubMed1999

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.

Amides↗
PubMed1999

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent.

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.

Apoproteins↗
PubMed1999

Upstream element of the sea urchin arylsulfatase gene serves as an insulator.

Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters. Here we report that a DNA fragment located in the upstream region of sea urchin, H. pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars enhancer when positioned between the enhancer and the target promoter, in an orientation dependent manner. The Ars insulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter. In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter. The insulation mechanism operates also unidirectionally in Drosophila. We also show that the efficiency of transformation of HeLa cells is enhanced when the integrated gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells. These results demonstrate that the mechanism of action of the insulator has been conserved throughout evolution.

Animals↗
PubMed1998

Arylsulfatase exists as non-enzymatic cell surface protein in sea urchin embryos.

The physiological role of arylsulfatase (Ars) and its function during development have yet to be satisfactorily defined in any species, though the proteins are widely distributed and the genes have been cloned from various organisms. Here we report the dual location of two types of Ars in sea urchin embryos. The majority of sea urchin Ars does not exhibit enzyme activity and is extracellularly distributed in aboral ectoderm cells (nonenzymatic Ars). Only a small portion has enzyme activity and is localized in lysosomal vesicles (enzymatic Ars). The elution pattern of Ars proteins processed by DEAE-cellulose or analytical gel-column chromatography reveals that although the molecular radius of enzymatic Ars differs from that of nonenzymatic Ars, they have the same charge. Furthermore, sedimentation analysis shows that purified Ars of sea urchin embryos is soluble in the absence of divalent cations but becomes insoluble in the presence of Ca2+ or Mg2+. Taken together, the present results suggest that non-enzymatic Ars is a new member of the cell surface component or extracellular matrix. It is possible that this cell surface Ars plays an important role in morphogenesis of sea urchin embryos.

Animals↗
PubMed1998

Effect of pressure on individual hydrogen bonds in proteins. Basic pancreatic trypsin inhibitor.

By performing two-dimensional 1H NMR measurements at 750 MHz at varying hydrostatic pressure (1-2000 bar) in an aqueous environment (90% 1H2O/10% 2H2O), we found that the signals of the peptide NH protons of basic pancreatic trypsin inhibitor (BPTI) in the folded state shift their positions linearly and reversibly with pressure. The strong tendency for low-field shifts of these protons indicates that most of the amide groups form hydrogen bonds either with carbonyls or with water and that these hydrogen bonds are shortened by pressure. The NH protons interacting favorably with solvent water tend to exhibit larger pressure-induced shifts than others, showing that the shift can be used as a diagnostic probe for the hydrogen bonding state of an NH group with water. Furthermore, we estimated shortening of individual H...O distances of the NH...O=C hydrogen bonds at 2000 bar on the basis of the empirical shift-distance correlation for BPTI. The estimated shortened distances varied considerably from site to site in the range of 0-0.11 A, larger in the turn but smaller in the interiors of secondary structures. These variations suggest that the volume fluctuation is heterogeneous within BPTI and that high-pressure NMR at high field can offer a unique opportunity for detecting microscopic structural fluctuation in proteins.

Aprotinin↗
PubMed1998

Structure and function of a sea urchin orthodenticle-related gene (HpOtx).

Two distinct types of orthodenticle-related proteins (HpOtxE/L) have been implicated as transcription activators of the aboral ectoderm-specific arylsulfatase (Ars) gene. Here, we describe the structure of HpOtx gene and present evidence that mRNAs of HpOtxE/L are transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing. By transactivation experiments, we have also demonstrated that HpOtxL activates the Ars promoter in the gastrula-stage embryo.

Animals↗
PubMed1998

The methanol-induced transition and the expanded helical conformation in hen lysozyme.

Methanol-induced conformational transitions of hen egg white lysozyme were investigated with a combined use of far- and near-UV CD and NMR spectroscopies, ANS binding and small-angle X-ray scattering. Addition of methanol induced no global change in the native conformation itself, but induced a transition from the native state to the denatured state which was highly cooperative, as shown by the coincidence of transition curves monitored by the far- and near-UV CD spectroscopy, by isodichroic points in the far- and near-UV CD spectra and by the concomitant disappearance of individual 1H NMR signals of the native state. The ANS binding experiments could detect no intermediate conformer similar to the molten globule state in the process of the methanol denaturation. However, at high concentration of methanol, e.g., 60% (v/v) methanol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far-UV CD spectroscopy, and had no specific tertiary structure, monitored both by near-UV CD and NMR spectroscopy. The radius of gyration in the H state, 24.9 angstroms, was significantly larger than that in the native state (15.7 angstroms). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea-denatured state, indicating a complete lack of globularity. Thus we conclude that the H state has a considerably expanded, flexible broken rod-like conformation which is clearly distinguishable from the "molten globule" state. The stability of both N and H states depends on pH and methanol concentration. Thus a phase diagram involving N and H was constructed.

Animals↗
PubMed1998

Quadriplegia caused by cervical hyperextension injury and intramedullary spinal cord tumour: a case report of autopsy.

A 68-year-old male had neck pain and weakness of the left upper extremity after a fall. MRI showed severe cervical canal stenosis and a high signal intensity of the spinal cord on T2-weighted images extending from the medulla oblongata to the C7 level. Neurological examination showed left hemiparesis, bilateral sensory disturbance and a neurogenic bladder. He underwent expansive laminoplasty 5 weeks later. After the operation his neurological deficit improved and 6 weeks later he left the hospital. However, his neurological conditions became worse (quadriparesis) and he was admitted as an emergency 3 weeks later. Although MRI showed decompression of the spinal cord, the area of high signal intensity on T2-weighted images had extended. Quadriparesis was progressive and he died of dyspnea. Autopsy showed the presence of the intramedullary spinal cord tumor (anaplastic astrocytoma; C1-Th4). We could not detect the intramedullary spinal cord tumour on MRI before surgery because of severe canal stenosis and the history of trauma. The high spinal intensity on T2-weighted images was thought to be oedema or myelomalacia. This case illustrates the difficulty of correctly interpreting MRI in patients with severe canal stenosis.

Aged↗
PubMed1998

High pressure NMR study of a small protein, gurmarin.

The effect of pressure on the structure of gurmarin, a globular, 35-residue protein from Gymnema sylvestre, was studied in aqueous environment (95% 1H2O/5% 2H2O, pH 2.0) with an on-line variable pressure NMR system operating at 750 MHz. Two-dimensional TOCSY and NOESY spectra were measured as functions of pressure between 1 and 2000 bar at 40 degrees C. Practically all the proton signals of gurmarin underwent some shifts with pressure, showing that the entire protein structure responds to, and is altered by, pressure. Most amide protons showed different degrees of low field shifts with pressure, namely 0-0.2 ppm with an average of 0.051 ppm at 2000 bar, showing that they are involved in hydrogen bonding and that these hydrogen bonds are shortened by pressure by different degrees. The tendency was also confirmed that the chemical shifts of the amide protons exposed to the solvent (water) are more sensitive to pressure than those internally hydrogen bonded with carbonyls. The pressure-induced shifts of the H alpha signals of the residues in the beta-sheet showed a negative correlation with the 'folding' shifts (difference between the shift at 1 bar and that of a random coil), suggesting that the main-chain torsion angles of the beta-sheet are slightly altered by pressure. Significant pressure-induced shifts were also observed for the side-chain protons (but no larger than 10% of the 'folding' shifts), demonstrating that the tertiary structure of gurmarin is also affected by pressure. Finally, the linearity of the pressure-induced shifts suggest that the compressibility of gurmarin is invariant in the pressure range between 1 and 2000 bar.

Amino Acid Sequence↗
PubMed1998

Proximal cis-regulatory elements of sea urchin arylsulfatase gene.

Expression of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, is regulated in spatially, as well as temporally, during development. To address the cis-regulatory elements involved in this regulation, we performed reporter assays using variously deleted or mutated promoter and regulatory elements of the HpArs gene, accompanied by gel mobility shift assay and foot printing. Results show that two regions, PU1 (-72 b.p. to -56 b.p.), which is similar to SpZ12-1 and/or Oct-1 motif, and the PD1 site (+133 b.p. to +142 b.p.), which is homologous to the binding sites of Rel family transcription factors and/or AGIE-BP1, are related to the regulation of expression of the HpArs gene. Furthermore, an HpArs enhancer element called C15, which is located 3 kb.p. downstream from the transcription start site, activates the HpArs promoter. We also report that the enhancer activity of the C15 fragment was mediated by elements, PU1 and PD1.

Animals↗