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Biomedical subjects

Y Fukada

Publications and source records attributed to Y Fukada.

At least 145 records · Page 8Linked to original sources
PubMed1989

Effect of chloride ion on the thermal decay process of the batho intermediate of iodopsin at low temperature.

The photochemical and the subsequent thermal behaviors of iodopsin (Cl(-)-bound form) and N-iodopsin (iodopsin whose Cl- was replaced by NO3-) in CHAPS-phosphatidylcholine (PC) were studied by low-temperature spectrophotometry. Irradiation of the iodopsin preparation at -185 degrees C produced a photo-steady-state mixture composed of iodopsin, bathoiodopsin, and isoiodopsin. Bathoiodopsin was thermally reverted to the original iodopsin. These results were almost the same as those reported previously [Yoshizawa, T., & Wald, G. (1967) Nature 214, 566-571] in which iodopsin was extracted with 2% digitonin. Therefore, photochemical and subsequent thermal behaviors of iodopsin were independent of the detergent to solubilize iodopsin. Irradiation of N-iodopsin at -185 degrees C produced the similar photo-steady-state mixture. However, N-bathoiodopsin was thermally converted to the next intermediate, presumably N-lumiiodopsin. These results suggest that the batho-lumi transition of iodopsin at low temperature is likely to be inhibited by the Cl- bound to the protein moiety of iodopsin, while at room temperature the Cl- bound to iodopsin could be released on the conversion process of batho- to lumiiodopsin.

Animals↗
PubMed1989

Purification of cone visual pigments from chicken retina.

A novel method for purification of chicken cone visual pigments was established by use of a 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate-phosphatidylcholine (CHAPS-PC) mixture. Outer segment membranes isolated from chicken retinas were extracted with 0.75% CHAPS supplemented with 1.0 mg/mL phosphatidylcholine (CHAPS-PC system). After the extract was diluted to 0.6% CHAPS, it was loaded on a concanavalin A-Sepharose column. Elution from the column with different concentrations of methyl alpha-mannoside yielded three fractions: the first was composed of chicken violet, blue, and red in roughly equal amounts, the second predominantly contained chicken red, and the third was rhodopsin with a small amount of chicken green, which was separated from rhodopsin by DEAE-Sepharose column chromatography. Since CHAPS has little absorbance at both ultraviolet and visible regions, we could demonstrate the absolute absorption spectra of chicken red (92%) and rhodopsin (greater than 96%) in these regions. The maximum of the difference spectrum between either chicken red or rhodopsin and its photoproduct (all-trans-retinal oxime plus opsin) was determined to be 571 or 503 nm, respectively. Although chicken green was contaminated with a small amount of rhodopsin having a similar spectral shape, the maximum of its difference spectrum was located at 508 nm by taking advantage of the difference in susceptibility against hydroxylamine between these pigments. Although chicken blue and chicken violet were minor pigments present in the first fraction from the concanavalin A column, their maxima in the difference spectra were determined to be at 455 and 425 nm, respectively, by a partial bleaching method.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Identification of a retina-specific MEKA protein as a 33 K protein.

A photoreceptor-specific MEKA protein was purified from bovine retinal soluble fraction. The purified sample was eluted as a single peak of 74 kDa protein from a Superose column, which was dissolved into three components, MEKA protein (32 kDa), beta-(36 kDa) and gamma-(10 kDa) subunits of transducin on a SDS-PAGE. From several lines of evidence, we concluded that MEKA protein is identical with a 33k phosphoprotein reported by Lee et al (1).

Animals↗
PubMed1989

Beta gamma-subunit of bovine transducin composed of two components with distinctive gamma-subunits.

During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.

Adenosine Triphosphate↗
PubMed1989

Chicken red-sensitive cone visual pigment retains a binding domain for transducin.

Iodopsin (a red-sensitive cone visual pigment) and rhodopsin (a rod pigment) were isolated from chicken retina. They were separately reconstituted into phosphatidylcholine liposomes and then mixed with rod transducin (T alpha and T beta gamma) purified from bovine retina. Iodopsin enhanced, only when irradiated, the binding of GppNHp to T alpha to a similar extent to irradiated rhodopsin. Furthermore, the binding of GppNHp to T alpha in the presence of a photobleaching intermediate of iodopsin preferably required T beta gamma-2 rather than T beta gamma-1, which is very similar in profile to that in the presence of the intermediate of rhodopsin (J. Biol. Chem., in press). These results indicate that the binding domain for transducin in iodopsin should closely resemble that in rhodopsin.

Animals↗
PubMed1989

Monoclonal antibodies to chicken iodopsin.

The protein moiety of chicken iodopsin, R-photopsin, was purified from the chicken retina using a sucrose flotation method followed by two steps of column chromatography. Apparent molecular weights of R-photopsin and scotopsin (the protein moiety of chicken rhodopsin), which was partly purified in the process of purification of R-photopsin, were estimated to be 34,000 and 36,000, respectively, by sodium docecylsulfate-polyacrylamide gel electrophoresis. Using the purified R-photopsin as an antigen, four kinds of hybridoma cells which secreted monoclonal antibodies specific for R-photopsin and iodopsin were prepared. The antibodies thus obtained reacted with neither other chicken cone visual pigments nor rhodopsin as analyzed by immunoblots and immunoprecipitation methods. All the monoclonal antibodies stained the majority of the cone outer segments in chicken retina, while an antiserum raised against cattle rhodopsin stained the rod outer segments as well as some cone outer segments in the retina.

Animals↗
PubMed1989

Underlying mechanisms of the response specificity of expansion/contraction and rotation cells in the dorsal part of the medial superior temporal area of the macaque monkey.

1. The dorsal part of medial superior temporal area (MST) has two unique types of visually responsive cells: 1) expansion/contraction cells, which selectively respond to either an expansion or a contraction; and 2) rotation cells, which selectively respond to either a clockwise or a counterclockwise rotation. In addition to selectivity for the mode of motion, both types of cells respond preferentially to movements over a wide field rather than over a small field. With the aim of understanding the underlying mechanisms of these selectivities, we carried out experiments on immobilized monkeys anesthetized with N2O. 2. Expansion/contraction and rotation of a pattern extending over a wide field contain three stimulus factors: 1) the spatial arrangement of different directions of movement, 2) the gradient in the speed of regional movement from the center to the periphery of the stimulus, and 3) the size change of texture components of the pattern in the expansion/contraction and the acceleration of movement of texture components toward the center of the stimulus in the rotation. The contribution of each factor to the activation of the cells was evaluated by comparing the response before and after removing the factor from the stimulus. The moving stimuli that lacked one or two of the factors were produced by the use of a cinematographic animation technique. 3. Withdrawal of the first factor, the spatial arrangement of different directions of movement, reduced the response of both Expansion/contraction and Rotation cells much more severely than either of the other two factors. We concluded that the first factor is far more important for activation than the other two. 4. These results are consistent with the model that Expansion/contraction and Rotation cells receive converging inputs from many directional cells with relatively small receptive fields in different parts of the visual field. Because MST receives strong fiber projections from MT, MT cells are candidates for the input cells. According to the model, if the convergence is organized so that the preferred directions of the input cells are arranged radially, the target cell will be an Expansion/contraction cell; if the input cells are arranged circularly, a Rotation cell will result.

Animals↗
PubMed1988

Analysis of discontinuity in visual contours in area 19 of the cat.

Previous ablation studies have suggested that area 19 of the cat plays an important role in pattern discrimination. To clarify the functional roles unique to area 19, we studied the receptive-field properties of cells in area 19 and compared them with those of cells in area 17. Recordings were made of anesthetized and immobilized animals. The majority (72%) of the cells in area 17 responded maximally to an elongated bar at a particular orientation, while they responded only weakly or not at all to a small spot (elongation-requiring cells). In contrast, more than half (63%) of the cells in area 19 showed a good response to a nonoriented small stimulus moving in any direction (dot-responsive cells). Two-thirds of the dot-responsive cells in area 19 failed to respond when the moving slit was elongated to more than some length in any orientation. These dot-responsive cells of the "inhibited-by-length" type responded strongly to the end of a long bar, and many of them also responded strongly to a break point in the middle of a long bar. We suggest that these dot-responsive cells of the "inhibited-by-length" type detect discontinuities in contours. Though they are in the minority, elongation-requiring cells constitute a considerable population (37%) in area 19, and dot-responsive and elongation-requiring cells from columnar patches in the same area. We conclude that, in contrast to area 17, whose main role is the decomposition of patterns into oriented contours, area 19 analyzes both orientation and discontinuities, with a strong bias towards the latter.

Animals↗
PubMed1986

Gain control mechanisms in X- and Y-type retinal ganglion cells of the cat.

A change in responsiveness caused by a spot of light (conditioning spot, CS; 3 sec in duration) presented within a central region of the receptive field of X- and Y-type retinal ganglion cells of the cat was investigated by measuring the magnitude of responses to another spot of light (test spot, TS; 50 msec in duration) which was juxtaposed with the CS within the same receptive field's central region. Responses to the TS were suppressed steadily during the on-phase of the CS as if it were divided by a certain value. This fact indicates that the gain of the center mechanism was changed by the CS presentation. The setting of the gain to a new level was rapid (within 100 msec after the onset or the cessation of the CS), and the magnitude of a gain change was not affected by the surround antagonism. These characteristics of the gain control were common to X- and Y-cells under both mesopic and scotopic levels of light adaptation.

Action Potentials↗
PubMed1986

Analysis of local and wide-field movements in the superior temporal visual areas of the macaque monkey.

The middle temporal (MT) and medial superior temporal (MST) areas of the macaque cortex have many cells that respond to straight movements in the frontoparallel plane with directional selectivity (D cells). We examined their responses to movements of a bar, of a wide dot pattern, and to combined movements of the two in anesthetized and immobilized animals. D cells in MT showed a wide variety in the strength of the inhibitory field surrounding the excitatory center field. Responses of SI+-type cells to a bar moving across the excitatory field were suppressed when a wide dot pattern moved over the surround field in the same direction and at the same speed as the bar. Inhibition was selective to the direction and speed of the surround movement, and the effective area for inhibition occupied a wide area, which expanded in all radial directions. Responses of SI- -type cells to a center bar movement were changed little by a conjoint movement over the surround field. Consequently, SI- -type cells responded to wide-field movement as well as to stimuli confined within the excitatory field. Although D cells in MST commonly had large excitatory fields, a proportion of them (Figure type) responded to bar movement much more strongly than to wide-field movement. Their responses to a bar movement were suppressed direction-selectively by a conjoint movement of a wide dot-pattern background. The effective area for inhibition coexisted with the excitatory field in these cells. MST cells of the Nonselective type responded comparably well to the two stimuli, and those of the Field type responded much more strongly to wide-field movement than to bar movement. It is thus suggested that MT cells of the SI+ type and MST cells of the Figure type can detect a difference between movements of an object and its wide background, whereas MST cells of the Field type can detect a conjoint movement of a wide field, neglecting the movements of a single object.

Acoustic Stimulation↗
PubMed1986

Integration of direction signals of image motion in the superior temporal sulcus of the macaque monkey.

Using anesthetized and paralyzed monkeys, we have studied the visual response properties of neurons in the cortical area surrounding the middle temporal area (MT) in the superior temporal sulcus (STS). Systematic electrode penetrations revealed that there is a functionally distinct region where three classes of directionally selective cells with large receptive fields cluster. This region is anteriorly adjoined to the dorsal two-thirds of MT, has a width of 4-5 mm mediolaterally, and therefore may correspond to the dorsal part of the medial superior temporal area (MST), which was previously defined as a MT-recipient zone. One class of cells responded to a straight movement of patterns in the frontoparallel plane with directional selectivity (D cells: 217/422, 51.4%). The second class of cells selectively responded to an expanding or contracting size change of patterns (S cells: 66/422, 15.7%). These cells responded neither to a change in width of a slit of any orientation or any length, nor to a change in brightness. The third class of cells responded only to a rotation of patterns in one direction (R cells: 58/422, 13.7%). A majority of these cells (41/58) responded to the clockwise or counterclockwise rotation of patterns in the frontoparallel plane (Rf cells), while the rest responded to a rotation of patterns in depth (Rd cells). We will suggest that these cells acquire the ability to discover whole events of visual motion--i.e., unidirectional straight movement, size change (radial movement), and rotation--by integrating elemental motion information extracted by MT cells. The receptive fields of D, S, and Rf cells can be constructed by converging signals of MT cells, the preferred directions of which are arranged in parallel (D cells), radially (S cells), and circularly (Rf cells). The receptive fields of Rd cells can be constructed, in turn, by the convergence of signals of S cells.

Animals↗
PubMed1985

Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassay.

Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung.

Amniotic Fluid↗
PubMed1985

Two-site "simultaneous" immunoassay with monoclonal antibodies for the determination of surfactant apoproteins in human amniotic fluid.

Monoclonal antibodies against human surfactant apoproteins were prepared, which recognized 37, 34, and 62 kilodalton proteins in human lung lavage fluid and amniotic fluid. Two-site "simultaneous" immunoassay for the surfactant apoproteins was developed using the monoclonal antibodies. The assay was capable of measuring 10-640 ng of the apoproteins per ml of human amniotic fluid. The immunoassay was used to quantitate the apoproteins in 59 amniotic fluid samples from 23 to 41 wk gestation. The concentration of the surfactant apoproteins at less than 30 wk gestation was very low (mean 0.84 micrograms/ml). It then increased 6.5-fold from 34 to 36 wk gestation and 15.5-fold at more than 37 wk gestation. The simultaneous immunoassay with the monoclonal antibodies presented herein seems to be ideal for clinical use because of its high specificity, sensitivity, rapidity, simplicity, and a continuous unlimited supply of the antibodies. The results in this study show that the clinical use of the two-site simultaneous immunoassay with monoclonal antibodies to pulmonary surfactant apoproteins can predict fetal lung maturity more precisely.

Amniotic Fluid↗
PubMed1984

Studies on structure and function of rhodopsin by use of cyclopentatrienylidene 11-cis-locked-rhodopsin.

The photochemical reaction of cyclopentatrienylidene 11-cis-locked-rhodopsin derived from cyclopentatrienylidene 11-cis-locked-retinal and cattle opsin was spectrophotometrically studied. The difference absorption spectrum between the cyclopentatrienylidene 11-cis-locked-rhodopsin and its retinal oxime had its maximum at 495 nm (P-495). Irradiation of P-495 at -196 degrees C with either blue light or orange light caused no spectral change, supporting the cis-trans isomerization hypothesis for formation of bathorhodopsin. Upon irradiation of P-495 at 0 degree C with orange light, however, its absorption spectrum shifted to a shorter wavelength owing to formation of a hypsochromic product. The difference absorption spectrum between this product (P-466) and its retinal oxime showed its maximum at 466 nm. Analysis of retinal isomers by high-performance liquid chromatography showed that this spectral shift was not accompanied by photoisomerization of the chromophore. P-466 could almost completely be photoconverted to the original pigment (P-495) by irradiation at 0 degree C with blue light with little formation of the other isomeric form of its chromophore. The alpha-band of the circular dichroism spectrum of P-495 was very small in comparison with that of rhodopsin, while that of P-466 was comparable to it. These facts suggest that P-495 has a planar conformation in the side chain of the chromophore and that P-466 has a twisted one, probably at the C8-C9 single bond. Cyclic-GMP phosphodiesterase in frog rod outer segment was activated by neither P-495 nor P-466. This result suggests that the isomerization of the retinylidene chromophore of rhodopsin is indispensable in the phototransduction process.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1983

Activation of phosphodiesterase by rhodopsin and its analogues.

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of beta-ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an "expanded" conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to -12 degrees C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1982

Activation of phosphodiesterase by chicken iodopsin.

Activation of guanosine 3',5'-cyclic monophosphate phosphodiesterase in outer-segment membrane of chicken retina was investigated. Irradiation of dark-adapted chicken outer segment membrane for bleaching of iodopsin increased the enzyme activity twice as much as that in the dark in the presence of GTP. Further irradiation of the sample for bleaching of rhodopsin in the membrane induced some additional activation of the enzyme. However, chicken iodopsin activated the enzyme in frog rod outer segment membrane without irradiation, while chicken rhodopsin did not. Irradiation of chicken iodopsin increased the enzyme activity twice as much as that in the dark.

3',5'-Cyclic-GMP Phosphodiesterases↗