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K Akasaka

Publications and source records attributed to K Akasaka.

At least 73 records · Page 4Linked to original sources
PubMed1998

Differential expression of sea urchin Otx isoform (hpOtxE and HpOtxL) mRNAs during early development.

Two distinct types of orthodenticle-related proteins (early type: HpOtxE, late type: HpOtxL) of the sea urchin, Hemicentrotus pulcherrimus, have been implicated as enhancer element binding factors of the aboral ectoderm-specific arylsulfatase (HpArs) gene. In order to understand the role of these isoforms during sea urchin development, we have isolated and characterized HpOtx gene. Here we describe the spatial expression patterns of HpOtxE and HpOtxL mRNAs and effects of overexpression of these mRNAs on embryogenesis. Whole-mount in situ hybridization using each isoform-specific probe reveals the complex and dynamic change of expression patterns among three germ layers. HpOtxE mRNA is maternally stored and exists apparently in a nonlocalized manner by the blastula stage. After hatching, HpOtxE transcripts are expressed predominantly in presumptive endoderm cells and gradually decrease during gastrulation. Signals for HpOtxL mRNA are intense at the vegetal half after hatching and subsequently, its expression is restricted to the micromere-derived cells. After primary mesenchyme cell (PMC) ingression, HpOtxL transcripts are localized at the vegetal plate and thereafter, concentrated primarily in ectoderm. Eggs injected with HpOtxE or HpOtxL mRNA develop into similar radialized structures without PMC ingression and gut invagination, whose oral-aboral axes are disrupted. Overexpression of HpOtxE induces accumulation of HpOtxL mRNA at the significantly earlier stages, though HpOtxL overexpression inhibits the accumulation of HpOtxE transcripts. Expression patterns of HpOtxE and HpOtxL in all three germ layers and dramatic morphological changes observed in the mRNA-injected embryos suggest that each HpOtx isoform has an important role in sea urchin embryogenesis.

Animals↗
PubMed1998

[Left atrial function and left atrial appendage flow velocity in hypertrophic cardiomyopathy: comparison of patients with and without paroxysmal atrial fibrillation].

The involvement of left atrial (LA) appendage flow velocity in reduced left atrial function was investigated in 24 patients with hypertrophic cardiomyopathy, who retained sinus rhythm at the examination. Patients were divided into 11 with a history of paroxysmal atrial fibrillation [PAf(+)] and 13 without such history [PAf(-)]. Transthoracic echocardiography was performed to evaluate LA fractional shortening (LA%FS) and mean velocity of circumferential LA fiber shortening (LAmVcf), as contractile functions of the left atrium at the phase of active atrial contraction. Transesophageal echocardiographic Doppler examination was performed in all patients to measure the LA appendage velocity. In all patients, significant positive correlations were observed between the LA appendage velocity and LA%FS (r = 0.50, p < 0.05) or LAmVcf (r = 0.82, p < 0.001). LAmVcf and LA appendage velocity in patients with paroxysmal fibrillation were significantly lower than in those without (0.84 +/- 0.15 vs 1.28 +/- 0.37 circ/sec, 44 +/- 12 vs 65 +/- 20 cm/sec, both p < 0.01), whereas LA diameter was greater in the former compared to the latter (45 +/- 5 vs 38 +/- 5 mm, p < 0.01). LAmVcf and LA appendage velocity were low in four patients with cerebral infarction or transient cerebral ischemic attack (LAmVcf < 1.0 circ/sec, LA appendage velocity < or = 40 cm/sec). Importantly, all these patients had a history of paroxysmal fibrillation. These results indicate that there is a close relationship between LA appendage velocity and LA contractile function in patients with hypertrophic cardiomyopathy with paroxysmal atrial fibrilation, and these patients have potential risk of cerebral infarction.

Adult↗
PubMed1997

Pressure-induced changes in the folded structure of lysozyme.

We demonstrate, for the first time in solution, that pressure induces changes in the overall folded structure of a protein (lysozyme). This was made possible by using a home-developed, on-line continuously variable pressure cell on a high resolution NMR spectrometer operating at 750 MHz. We could follow pressure-induced diamagnetic chemical shifts of more than 26 protons of lysozyme at variable pressure in the range of 1 to 2000 bar. The results indicate that the main effect of the pressure is a compaction of the hydrophobic core part of the protein consisting of bulky side-chains. The technique introduced here provides a general method with which one can probe microscopic internal flexibility of a protein in solution.

Animals↗
PubMed1997

Identification of MAPKAPK homolog (MAPKAPK-4) as a myosin II regulatory light-chain kinase in sea urchin egg extracts.

We identified and cloned a homolog of mammalian mitogen-activated protein kinase-activated protein kinase (MAPKAPK)-2 and -3 from sea urchin, Hemicentrotus pulcherrimus. The obtained cDNA clone was composed of 350 amino acid residues which contain MAPK phosphorylation sites and the bipartite nuclear localization signal sites in its C-terminal domain. The clone showed 65.4 and 66.7% amino acid residue identity to human MAPKAPK-2 and -3, respectively. Phylogenetic analysis revealed that the homolog can be classified into a distinct group of MAPKAPK and, therefore, the identified homolog was designated as MAPKAPK-4. Biochemical characterization was performed using recombinant glutathione S-transferase (GST)-MAPKAPK-4 fusion protein. The protein kinase activity of GST-MAPKAPK-4 was activated by MAPK and this enabled the kinase to phosphorylate both glycogen synthase N-terminal peptide and the regulatory light chain of myosin II in vitro. Northern blot analysis showed that MAPKAPK-4 was expressed throughout the development of sea urchin embryos. These observations suggest that MAPKAPK-4 may play an important role in the regulation of myosin II activity during the development of sea urchin.

Amino Acid Sequence↗
PubMed1997

Two isoforms of orthodenticle-related proteins (HpOtx) bind to the enhancer element of sea urchin arylsulfatase gene.

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene, expressed specifically in aboral ectoderm, contains a 229-bp enhancer in its first intron that is required for the activation of HpArs gene expression. Deletion analysis shows that a tandem repeat of orthodenticle-related protein (Otx) binding sites are responsible for the activity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift assays, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at the blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stage. Two distinct types of HpOtx cDNA clones have been isolated from cDNA libraries of unfertilized eggs and gastrulae. Nucleotide sequences of the homeobox and downstream regions are well conserved in the two types of HpOtx cDNAs, while the region upstream from the homeobox has different nucleotide sequences. By genomic Southern blot analysis, only a single copy of HpOtx gene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Results from Northern blot analysis confirm the presence of two types of HpOtx transcripts. Transcriptional regulation of the HpArs gene may, in part, be carried out through switching of Otx isoforms.

Amino Acid Sequence↗
PubMed1997

Urea-induced conformational changes in cold- and heat-denatured states of a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.

Bacterial Proteins↗
PubMed1997

Oral-aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore.

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3 h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral-aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral-aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oral-aboral axis during sea urchin development.

Animals↗
PubMed1997

A cis-regulatory element within the 5' flanking region of arylsulfatase gene of sea urchin, Hemicentrotus pulcherrimus.

The 5' flanking region of the sea urchin Hemicentrotus pulcherrimus arylsulfatase (Ars) gene was scanned to define cis-regulatory elements required for proper expression congruent to that of the endogenous gene. The region between -100 bp and +38 bp from the transcription start site contains minimum information required for temporal initiation of transcription of the Ars gene. Progressive deletion analysis of Ars-luciferase reporter constructs containing the Ars sequence from -3484 bp to +38 bp suggests the existence of several cis-regulatory elements within this region. Results from luciferase assays of internal deletion mutants show strong enhancer activity detected within the sequence from -194 bp to -144 bp. By gel mobility shift assay, we have identified a nuclear factor that interacts sequence-specifically with this 50 bp region, and appears in a developmental stage-specific manner. Further deletion analysis determined that the enhancer activity lies within a 22 bp sequence between -186 bp and -164 bp.

Animals↗
PubMed1997

An in vitro 1H-MRS model of oncogene transfection. The spectral feature of c-erbB-2 and c-Ha-ras transfected NIH3T3 fibroblast cells.

PURPOSE: Malignancy is an abnormality of cell division and differentiation based on abnormal expression of oncogenes. This note describes the in vitro 1H-NMR spectral features of oncogene-transfected NIH3T3 fibroblast cells compared to non-transfected cells. MATERIAL AND METHODS: 1H-NMR spectra of cultured NIH3T3 cells and c-erbB-2 or c-Ha-ras gene-transfected cells were obtained by 400 MHz high resolution NMR. The peaks were assigned by 2D HOHAHA spectra of the cell suspension and the spectral changes were evaluated in 1D and 1D differential spectra. RESULTS: The 1H spectra obtained from both transfected cell lines were broadened over all peaks, suggesting reduced mobility in plasma membrane lipid molecules. No other differential spectra for characterizing metabolic change was detected. CONCLUSION: Broadened 1H spectra observed after c-erbB-2 or c-Ha-ras transfection suggest changes of plasma membrane viscosity, which may be related to the oncogene expression.

3T3 Cells↗
PubMed1997

EMG power spectrum and integrated EMG of ankle plantarflexors during stepwise and ramp contractions.

The aim of this study was to investigate whether the median frequencies (MF) of the electromyogram (EMG) and the integrated EMG (IEMG) of histochemically differentiated ankle plantarflexors, the gastrocnemius and soleus, were force dependent. Bipolar intramuscular wire electrodes were used to measure EMG of the soleus (SO), medial head of gastrocnemius (GM), and lateral head of gastrocnemius (GL) during ramp (single ongoing contractions) with the force increasing linearly from 0 to 100% of maximum voluntary contraction (MVC) and stepwise (steady force levels) ankle plantarflexion at 10, 20, 30, 40, 60, and 80% MVC. EMG and force were measured simultaneously. Power spectral analysis of these signals was performed to calculate MF on 1024-point by fast Fourier transform (FFT) technique. IEMG value of each muscle was also obtained at the same levels of force. While IEMG of three heads of triceps surae in both stepwise and ramp contractions increased significantly with increasing force, MF values of GL during stepwise contraction increased significantly (20, 40, 60, 80% MVC). These results suggest that the sensitivity of EMG power spectrum might be influenced by the proportion of fast twitch muscle fibers, which histochemically corresponds to type II fibers.

Adult↗
PubMed1997

Cloning of cyclin E cDNA of the sea urchin, Hemicentrotus pulcherrimus.

cDNA encoding maternal cyclin E (HpCycE) has been cloned from the oocyte cDNA library of the sea urchin, Hemicentrotus pulcherrimus, by differential screening with a cDNA probe covering the total poly(A)+ RNAs of 16 cell-stage embryos and gastrulae. In this communication we describe similarity of amino acid sequences between HpCycE and those of cyclin E from other organisms and maternal origin of HpCycE. The amino acid sequence deduced from the nucleotide sequence of HpCycE cDNA is highly similar to those of human, rat, chicken, Xenopus, zebrafish and Drosophila, while its similarity to other cyclins is much lower. A gene for HpCycE exists as a single copy in the genome of H. pulcherrimus. Northern blotting revealed that the mRNA for HpCycE is maintained at a high level up to the morula stage and thereafter declines.

Animals↗
PubMed1997

[Association between sleep apnea syndrome and coronary artery disease].

Patients with sleep apnea syndrome often suffer from cardiovascular disease, but the incidence of coronary artery disease (CAD) with coronary spasm in these patients is not known. In the present study, 14 of 37 men with sleep apnea syndrome diagnosed by all-night polysomnography were suspected to also have CAD, based on the results of non-invasive clinical examinations. Coronary angiography confirmed the diagnosis of CAD either organic stenosis or coronary spasm in 8 of the 14 patients. Those 8 did not differ significantly from the 21 patients without CAD, with regard to coronary risk factors or to the severity of their sleep apnea (apnea index, 4% desaturation ratio, and nadir of SaO2). Eleven patients received intracoronary injections of acetylcholine, which induced coronary spasm in 4 (36.4%) and coronary contraction in 2 (18.2%). Coronary spasm was induced in 4 of the patients with CAD (50.0%). Although the pathophysiologic link between sleep apnea syndrome and CAD is still unclear, these results suggest that patients with this syndrome frequently suffer from CAD, particularly from coronary spasm.

Adult↗
PubMed1996

The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering.

Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering. At pH 3.0 and 25 degrees C, two methanol-induced non-native states were characterized. First, addition of methanol up to 25% (v/v) induced a compact denatured conformer (I(M)). Further addition of methanol transformed this I(M) state into the expanded and highly helical denatured state (H). The existence of the I(M) state was shown by the discrepancy in transition curves obtained from the ellipticity at 222 nm, the ellipticity at 282 nm, the tryptophan fluorescence monitored at 350 nm and the native peak intensity of the (1)H NMR spectrum. These CD, fluorescence and NMR results showed that the I(M) state has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state. The radius of gyration of the I(M) state, 17.7 angstroms, obtained from the Guinier plot of the small-angle X-ray scattering data was significantly smaller than that of the acid-denatured state (30.1 angstroms) and was closer to that of the native state (14.6 angstroms), showing that the I(M) state is compact. The Kratky plot for the I(M) state exhibited a bell-shaped profile, indicating a globular conformation. These structural features indicate that the structure of the I(M) state is quite similar to that of the anion-induced molten globule state of this protein. Furthermore the alcohol-denatured state (H) of cyt C in 60% (v/v) methanol was structurally characterized. Though the H state had a helical content much higher than the native state monitored by far-UV CD spectroscopy, the radius of gyration, 31.7 angstroms, was similar to that of the acid-denatured state, showing that this H state is an expanded denatured state. The Kratky plot for the H state did not show a clear peak, indicating a chain-like conformation. Thus we conclude that the H state has an expanded and chain-like conformation with a high helical content. Finally, we constructed a phase diagram of cyt c involving the native, I(M), acid-denatured and H states against pH and the methanol concentration. The result indicates that the I(M) state is found in the pH range from 2.5 to at least 4.5 with a pH-dependent optimum methanol concentration of 10 to 40%.

Circular Dichroism↗
PubMed1996

Fluorimetric determination of diarrhetic shellfish toxins in scallops and mussels by high-performance liquid chromatography.

The fluorimetric determination of okadaic acid (OA) and dinophysistoxin-1 (DTX-1), the principal toxins of diarrhetic poisoning, is reported. The digestive glands of mussels or scallops were homogenized with 2-propanol. OA and DTX-1 were extracted from the homogenate, with hexane-ethyl acetate and labelled with 2,3-(anthracenedicarboximido)ethyl trifluoromethanesulfonate in dry acetonitrile. After cleaning up by passage through a short silica gel column, the fluorescent derivatives were determined by HPLC. The derivatives were at first separated on a Develosil Ph-5 column, and only the target fraction obtained was introduced into a Develosil ODS K-5 column by a valve-switching device. Both toxins were determined in the range 2.5-500 pg, and the detection limits were 0.8 pg (OA) and 1.3 pg (DTX-1) with a signal-to-noise ratio of 3.

Animals↗
PubMed1996

Differential structural requirements for interaction of Ras protein with its distinct downstream effectors.

Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

Adenylyl Cyclases↗
PubMed1996

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state.

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10)s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8)s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8)s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10)s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.

Bacterial Proteins↗
PubMed1996

Spatial expression of a forkhead homologue in the sea urchin embryo.

Echinoderms are the sister group of the chordates and hemichordates within the deuterostomes. They lack a notochord or any structures obviously homologous with it. To gain insight into developmental mechanisms important in the origin and early evolution of chordates, we investigated sea urchin homologues of chordate genes that are implicated in notochord formation, viz. Brachyury and HNF-3 beta. Here we report the pattern of expression of a sea urchin orthologue of forkhead, Hphnf3 which is present as a single copy per haploid genome. An Hphnf3 transcript of 3.0 kb was first detected at the swimming blastula stage, accumulated maximally at the gastrula and prism-embryo stages, and decreased at the pluteus-larva stage. In situ hybridization signals were found in cells of the vegetal plate of the swimming blastula. During gastrulation, intense staining was evident in the cells surrounding the blastopore, whereas weak staining was detected in the invaginating archenteron. At the prism-embryo stage, the entire archenteron stained intensely; then, at pluteus stage, the larva staining decreased in intensity. The forkhead and Brachyury genes begin to be expressed almost simultaneously in sea urchin embryos, in the vegetal plate at the late blastula stage. After the onset of gastrulation, however, Hphnf3 is expressed in the posterior part of the archenteron, whereas the Brachyury orthologue, HpTa, is expressed in the secondary mesenchyme founder cells, which occupy the anterior tip of archenteron. Hphnf3 may contribute to specification of embryonic cells as archenteron, and the role of HpTa may be directed towards specification of mesodermal founder cells. Except for the basal character of expression in endoderm and endomesoderm, these transcription factors are clearly utilized differently in chordates.

Amino Acid Sequence↗