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Biomedical subjects

A Ikai

Publications and source records attributed to A Ikai.

At least 19 recordsLinked to original sources
PubMed2001

Accumulation of plant galactolipid affects cell morphology of Escherichia coli.

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.

Escherichia coli↗
PubMed2001

Force measurement and inhibitor binding assay of monomer and engineered dimer of bovine carbonic anhydrase B.

We applied atomic force microscopy (AFM) to study the intramolecular mechanics of the globular protein molecule, bovine carbonic anhydrase B. The immobilized protein on an amino-functionalized silicon wafer was pulled from its N- and C-termini after being covalently cross-linked to the AFM tip, and the relationship between the tensile force applied on the protein and its extension was recorded. The native enzyme (having 261 residues with two Cys added at its ends, and in a theoretical stretching length of 96 nm) was extended only to 13 +/- 2 nm under physiological conditions before disruption of the covalent cross-linking system. Contrary to the above observation, an engineered dimer was extended to about 110 nm even in the absence of the denaturant. The difference was ascribed to the presence or presumed absence of a "knot" structure at the C-terminal end of the two forms, respectively. When a specific inhibitor was added to the experimental solution, native monomers (sp activity = 88% of the wild type enzyme) were extended to 28 +/- 4 nm, whereas dimers (sp activity = 46%) were extended to about 56 +/- 3 nm, suggesting that both monomeric units in the dimer could bind inhibitor molecules, which was further corroborated by a titration experiment using a fluorescent inhibitor. Thus, one of the monomeric units in the engineered dimer was concluded to be enzymatically inactive but capable of binding inhibitors.

Animals↗
PubMed2001

Compact, reliable ventricular assist device as a bridge to recovery or for semipermanent use.

OBJECTIVES: The magnetically suspended centrifugal pump has gained attention as an implantable ventricular assist device for long-term use. We report recovery-oriented pump operation and results of a chronic animal experiment. METHODS: In an acute experiment in 8 sheep having microspheres injected to induce heart failure, left ventricular assist was implemented by 2 inflow cannulas, 1 each in the left atrium and left ventricle. The pressure-volume loop of the left ventricle and myocardial oxygen consumption were measured varying the assist rate. The chronic animal experiment used 10 sheep whose native heart was kept intact. Continuous hemodynamic monitoring and periodic blood sampling were conducted. RESULTS: In the acute study, myocardial oxygen consumption decreased proportionally with increasing assist rate in left atrial drainage, but was significantly less at a 100% assist rate in left ventricular drainage. External work in left ventricular drainage did not decrease until a 75% assist rate, suggesting that the left ventricle shape and size were maintained despite decreased myocardial oxygen consumption. In the chronic experiment, the pumping duration was 14 to 248 days. No thrombi or emboli in the pump or any major organ were found in sacrificed sheep after living more than 100 days. Hepatic and renal function were within an almost normal range throughout the experiment. CONCLUSION: Left ventricular blood drainage effectively reduced oxygen consumption, maintaining the shape of the left ventricle. The magnetically suspended centrifugal pump is suitable for recovery of a failing heart or semipermanent use.

Animals↗
PubMed2001

Sex differences in the hemoglobin oxygenation state of the resting healthy human masseter muscle.

AIMS: To determine whether sex differences exist in tissue oxygen saturation (StO2) and the hemoglobin (Hb) oxygenation state of the resting human masseter muscle. METHODS: Near-infrared spectroscopy (NIRS) was used to measure StO2 and Hb oxygenation state in 20 healthy adult volunteers (10 women and 10 men). To determine the measurement range and reliability of the NIRS recording probe, the probe was set up on 12 layers of white acrylic resin plate, each 3 mm thick. Total hemoglobin levels were measured while a red vinyl resin plate, 1 mm thick, was inserted in turn between each of the 12 layers. Distances from the skin surface to the lateral surface (S-L) and to the medial surface (S-M) of the right masseter at the middle portion of the masseter were measured on T1-weighted magnetic resonance images (repetition time 500 ms, echo time 23 ms). Thickness of the masseter was calculated by subtraction [(S-M)--(S-L)]. For the study of Hb oxygenation state, the probe was positioned at the same position on the skin surface at the mandibular postural (rest) position. RESULTS: The measurement range of the NIRS probe was from 9 to 21 mm under the skin, and the reliability of the probe was judged by intra- and inter-class correlation coefficients. There was no sex difference in S-L and the thickness of the masseter; the means of S-L and masseter thickness were 9.3 mm and 15.5 mm in men and 9.8 mm and 14.3 mm in women, respectively. Except for StO2 values, there were significant sex differences in the Hb oxygenation parameters, with the mean values in the men being approximately twice those in the women. CONCLUSION: These results provide evidence that a sex difference in the Hb oxygenation state may exist in the masseter muscle of normal healthy subjects.

Adult↗
PubMed2000

Novel method to enhance sternal healing after harvesting bilateral internal thoracic arteries with use of basic fibroblast growth factor.

BACKGROUND: Poor healing of the sternum often limits the use of bilateral internal thoracic arteries (BITAs) in coronary bypass surgery, especially for diabetic patients. We have reported that basic fibroblast growth factor (bFGF) enhanced regeneration of the skull. This study was designed to evaluate the effects of topical use of bFGF on sternal healing after removing the BITAs. METHODS AND RESULTS: Forty-five Wistar rats were subjected to median sternotomy and were divided into 3 groups: 15 had the BITAs removed and had a bFGF sheet applied on the posterior table of the sternum (group A), 15 had just the BITAs removed (group B), and 15 had intact BITAs (group C). Five and 10 rats were euthanized 2 and 4 weeks after surgery, respectively, in all 3 groups. Peristernal blood flow, measured with use of a noncontact laser flowmeter, decreased after removal of the BITAs (P:<0.001). Four weeks after the surgery, PBF markedly increased only in group A (9.7+/-1.2, 6.5+/-0.6, and 8.2+/-0.5 mL x min(-1) x 100 g(-1) for groups A, B, and C, respectively; P:<0.01 by ANOVA). Four weeks after surgery, the following findings were obtained only in group A: (1) nearly completely healed sternum filled with regenerated bone tissue, (2) marked angiogenesis around the sternum, and (3) osteoblasts in an active form around the edge of the sternum. CONCLUSIONS: The results suggest that use of the bFGF sheet offset the sternal ischemia and accelerated sternal healing. This method may help to decrease sternal necrosis in high-risk patients or allow extended use of BITAs in coronary bypass surgery.

Administration, Topical↗
PubMed2000

Dynamic measurement of single protein's mechanical properties.

Dimerized (tandemly repeated) protein was constructed, and the stretching force during the unfolding of the single protein molecule was measured using an atomic force microscope. In quasistatic measurements using normal force-distance curve measurements, each monomer unit was unfolded step by step. To elucidate the conformational state at each extension length, we measured the relax-stress response of the protein using short stroke sinusoidal movements of the sample stage. This allowed us to investigate the dynamic response of the protein repeatedly without full stretching or rupturing. Although the protein molecule responded in-phase to the applied movement in most cases, we found a novel out-of-phase response around the stretching length where the second monomer unit unfolded. Applying the spring constant measured in the quasistatic experiment, the out-of-phase response was reproduced in the simple calculation, which suggested the folding and the unfolding at the second monomer unit were taking place repeatedly during the relax-stress response measurement.

Animals↗
PubMed2000

Spring mechanics of alpha-helical polypeptide.

To design protein- and polymer-based micro-machineries, it is important to understand the mechanical properties of basic structural elements such as the alpha-helix of polypeptides. We employed the force measurement mode of an atomic force microscope (AFM) to investigate the spring mechanics of poly-L-glutamic acid (PGA) in its helical and randomly coiled states. After covalently anchoring the polypeptide between a silicon substrate and an AFM tip, the force required to stretch the polymer was measured. The results indicated that PGA in its helical conformation could be stretched almost fully with a continuous increase in the stretching force, suggesting that it can be used as a reliable coil-spring in the future design of spring-loaded molecular machineries.

Biomechanical Phenomena↗
PubMed1999

The distribution of sugar chains on the vomeronasal epithelium observed with an atomic force microscope.

The distribution of sugar chains on tissue sections of the rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined with an atomic force microscope (AFM). AFM tips were modified with a lectin, Vicia villosa agglutinin, which recognizes terminal N-acetyl-D-galactosamine (GalNAc). When a modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample surface were observed. The final rupture force was calculated to be approximately 50 pN based on the spring constant of the AFM cantilever. Distribution patterns of sugar chains obtained from the force mapping image were very similar to those observed using fluorescence-labeled lectin staining. AFM also revealed distribution patterns of sugar chains at a higher resolution than those obtained with fluorescence microscopy. Most of the adhesive interactions disappeared when the scanning solution contained 1 mM GaINAc. The adhesive interactions were restored by removing the sugar from the solution. Findings suggest that the adhesion force observed are related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium.

Acetylgalactosamine↗
PubMed1999

Continual neurogenesis of vomeronasal neurons in vitro.

We developed a culture system of vomeronasal neurons in which continuous degeneration and regeneration of axon bundles were observed. Partially dissociated vomeronasal cells from rat embryonic day 15 were grown in culture and formed a miniature vomeronasal-like epithelium. We called these structures vomeronasal pockets. They contained both vomeronasal neurons and supporting cells. They formed a spherical structure with a central cavity where microvilli protruded from supporting cells. Mature vomeronasal neurons with well-developed microvilli were not observed in the vomeronasal pocket. The time period between degeneration of axon bundles and the next was about 2 weeks. When vomeronasal pockets were incubated with 5 microgram/mL aphidicolin, an inhibitor of cell division, regeneration of axon bundles was not observed after degeneration. These results suggest that vomeronasal neurons in culture undergo continuous regeneration but do not fully mature. In this culture system, vomeronasal pockets survived for over 1 year.

Animals↗
PubMed1999

[The repair of congenital coronary artery fistula in the neonatal period].

We operated on 10 cases diagnosed as congenital coronary artery fistula, including 2 critical neonates. Such neonates needed special care for perfusion pressure drop at the beginning of cardiopulmonary bypass and imperfect delivery of cardioplegia. This phenomenon is likely to depend on the shunt size of a fistula, that is much larger in such neonates than in older patients who could be operated on electively. It was helpful in the situation to crossclamp the main PA, compress the fistula and crossclamp the aorta quickly for a standstill. We prefer the closure of a fistula in the recipient chamber especially when an important area is supplied distally to the origin of the fistula. However, if a ventricle is a recipient chamber, the closure through coronary incision should be done as the neonates in our series. There were two hospital deaths due to vascular leakage syndrome and hypoxia, respectively. Aortic regurgitation (AR) got worse in two after the operation. They were found to have more dilatation and distortion in the Valsalva's sinus which appeared to affect the aortic annulus to some extent. It is likely due to long-standing larger shunt. Contrary to them, the patient operated on during neonatal period was followed by no increase of AR, though she had the largest shunt of our series. We have an impression it could be prevented by earlier operation.

Aortic Valve Insufficiency↗
PubMed1998

Retrieval and amplification of single-copy genomic DNA from a nanometer region of chromosomes: a new and potential application of atomic force microscopy in genomic research.

Using a modified tip of the atomic force microscope (AFM), we harvested several strands of genomic DNA from a nanometer region of mouse chromosomes. We have also co-developed a random PCR method to amplify the recovered genomic DNA, in which a single DNA molecule of several kilobasepairs could be amplified efficiently. A subsequent fluorescence in situ hybridization (FISH) indicated that the amplified DNA originally came from the tip-manipulated regions of mouse chromosomes. Several fragments containing unique sequences were identified using Southern hybridization after subcloning the PCR products into pUC18 plasmid. The present results showed a potential application of AFM to genomic analysis.

3T3 Cells↗
PubMed1998

A new system for the measurement of electrogenicity produced by ion pumps using a thin polymer film: examination of wild type bacteriorhodopsin and the D96N mutant over a wide pH range.

We developed a new assay system for the measurement of capacitive electric currents generated by ion pumps using the thin polymer film 'Lumirror' (Toray Co., Japan). This system enables us to examine the electrogenicity of ion pumps over a wide range of experimental conditions with high reproducibility due to the mechanical and chemical stability, the high electric resistance and the high electric capacitance of the thin polymer film. Using this method, we examined the photoelectric response of wild type bacteriorhodopsin and its D96N mutant over a wide pH range (2.8-10.0). The results were explained in terms of the affinities of the proton binding sites for translocated protons. A possibility that the direction of the proton transfer from the Schiff base was influenced by the protonation/deprotonation state of the surrounding proton binding sites was suggested. We also found that this film can be used as a substrate for atomic force microscopy (AFM) samples and hence the active purple membrane was observed with AFM.

Bacteriorhodopsins↗
PubMed1998

Atomic force microscopy of histological sections using a new electron beam etching method.

In order to examine histological sections of the rat vomeronasal epithelium with the atomic force microscope (AFM), we developed an electron beam etching method that improves the resolution of AFM images. This method results in AFM images comparable to those obtained with the transmission electron microscope (TEM). Ultrathin tissue sections embedded in epoxy resin were observed before and after the treatment with electron beam radiation. Before electron beam treatment, epithelial structures such as the microvilli surface, dendritic processes, the supporting cell layers and the neuronal cell layers were all visible using the AFM. However, only a few subcellular structures could also be resolved. The AFM images were not as clear as those obtained with the TEM. After electron beam treatment, however, the resolution of AFM images was greatly improved. Most of the subcellular structures observed in TEM images, including the inner membrane of mitochondria, ciliary-structure precursor body, junctional complexes between the neurons and supporting cells, and individual microvilli were now visible in the AFM images. The electron beam treatment appeared to melt the embedding resin, bringing subcellular structures into high relief. The result of this study suggests that electron beam etching of histological samples may provide a new method for the study of subcellular structure using the AFM.

Animals↗
PubMed1997

Mapping cell wall polysaccharides of living microbial cells using atomic force microscopy.

Functionalized atomic force microscope tips were used to sense specific forces of interaction between ligand-receptor pairs and to map the positions of polysaccharides on a living microbial cell surface. Gold-coated tips were functionalized with concanavalin A using a cross-linker with a spacer arm of 15.6 A. It was possible to measure the binding force between concanavalin A and mannan polymers on the yeast (Saccharomyces cerevisiae) cell surface. This force ranged from 75 to 200 pN. The shape of the force curve indicated that the polymers were pulled away from the cell surface for a fairly long distance that sometimes reached several hundred nanometres. The distribution of mannan on the cell surface was mapped by carrying out the force measurement in the force volume mode of atomic force microscopy (AFM). During the measurement, the maximum cantilever deflection after contact between the tip and the sample was kept constant at 10 nm using trigger mode to keep the pressing force on the sample surface as gently as possible at a force of 180 pN. This regime was used to minimize the non-specific adhesion between the tip and the cell surface. Specific molecular recognition events took place on specific areas of the cell surface that could be interpreted as reflecting a non-uniform distribution of mannan on the cell surface.

Cell Membrane↗
PubMed1997

Morphologic study of the mandibular fossa and the eminence of the temporomandibular joint in relation to the facial structures.

To clarify the relationship between the temporal component of the temporomandibular joint and facial bone structure, we measured anthropologic craniometric points and the structure of the mandibular fossa, including the eminence in 33 Japanese male dry skulls, with a three-dimensional apparatus. The length from the articular tubercle to the medial glenoid tubercle is correlated with skeletal variables such as the length of the fossae, the width of the masseter origin, and the mandibular length. Therefore it appears that the frontal dimension is related to the facial development, which is attributable to bite force. Goniometrically, the angle between the line deepest point of the fossa--the midpoint of the eminence and the Frankfort horizontal plane (middle angle)--was negatively correlated only with the ANB angle, suggesting that a steeper middle angle of the eminence is related to a retrusive maxilla or protrusive mandible.

Adolescent↗