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Biomedical subjects

E Katayama

Publications and source records attributed to E Katayama.

At least 55 records · Page 3Linked to original sources
PubMed1989

The effects of various nucleotides on the structure of actin-attached myosin subfragment-1 studied by quick-freeze deep-etch electron microscopy.

Stereo electron microscopy of negatively stained images showed that myosin heads in acto-subfragment-1 (S1) covalently cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide were predominantly short and round when ATP was added, in contrast to their uniform tilted appearance in the rigor state. As an attempt to exclude molecules which were actually dissociated but still tethered to actin by artificial cross-links, quick-freeze deep-etch electron microscopy was coupled with the mica flake method to observe uncross-linked native acto-S1 in the presence of ATP. To maintain the low affinity S1 associated to actin in the presence of ATP, a high concentration of acto-S1 was applied to mica flakes whose absorption had been chemically modified. The image of acto-S1 with added ATP agreed well with the expected time-course of reversible dissociation and reassociation, confirming the applicability of this approach to examination of the structural changes of acto-S1. S1 molecules attached to F-actin under rigor conditions or in the presence of ADP were elongated, with the long axis tilted to F-actin. Actin-attached S1 became short and round upon addition of ATP or ADP-inorganic vanadate. Adenyl-5'-yl imidodiphosphate and inorganic pyrophosphate each partially dissociated S1 from actin, as expected.

Actins↗
PubMed1989

Assignment of the positions of chymotryptic fragments and cysteinyl groups in the primary structure of caldesmon in relation to a conformational change.

Caldesmon is universally associated with smooth muscle thin filaments, and reportedly interacts with actin, calmodulin, tropomyosin, and myosin. I attempted to determine the positions of the chymotryptic fragments which have been used to study the sites of such interactions in its primary structure. Such assignment, combined with the accumulated data of fragment studies, made it possible to clarify the functional domain organization of the caldesmon molecule. Using a specific cleavage method involving nitrothiocyanobenzoate, I also determined the number and locations of cysteinyl residues in the amino-acid sequence. Two cysteinyl groups thus determined were located close to the ends and almost at the same distance apart from the N- and C-termini of the whole molecule, respectively. Taking these locations and the extraordinary sensitivity to oxidation into consideration, I could reasonably elucidate the origin of the controversial data and the interpretation so far reported from various laboratories, concerning the length and conformation of the native molecule. Caldesmon might be folded in solution and its contour length could be almost double the accepted value, which was hydrodynamically estimated.

Amino Acid Sequence↗
PubMed1989

Characteristics of rat kidney dopamine receptors and the effects of renal denervation and dopamine infusion on these receptors.

The characteristics of dopamine receptors, as well as the effects of denervation and dopamine infusion on dopamine receptors were studied using the radiolabeled receptor assay of [3H]-spiperone on rat kidney membrane preparations. The rat renal cortex was found to have a single class of [3H]-spiperone binding sites with a dissociation constant (Kd) of 13.5 +/- 2.2 nM. However, neither sulpiride nor serotonin strongly interacted with [3H]-spiperone binding, suggesting that DA1 receptors were predominant in the rat renal cortex. Denervation was performed by surgically stripping the nerves from the renal artery and coating them with 10% phenol. Chronic denervation had no significant effect on the affinity or maximum binding capacity of the renal dopamine receptors, although diuresis and the disappearance of catecholamine fluorophores in the denervated rats were observed. Chronic infusion of dopamine was performed using an osmotic minipump, resulting in a decrease in the number of rat kidney dopamine receptors. These results suggest that the dopamine receptor subtype in the renal cortex was mainly DA1, and that the major source of dopamine which affected the dopamine receptors in the rat kidney was not the nerve ending, but rather the circulation.

Animals↗
PubMed1989

Localization of alpha 1-adrenoceptor in the rat kidney: in vitro autoradiography using [3H]-bunazosin.

We studied the precise localization of alpha 1-adrenoceptor in the rat kidney using in vitro macro- and micro-autoradiography (ARG) of [3H]-bunazosin. The slide-mounted nonfixed frozen sections (10 micron) of kidney were incubated with [3H]-bunazosin (5 nM) at 24 degrees C for 45 min, and then applied to macro- and micro-ARG. The macro-ARG revealed that the bunazosin binding sites were present mainly in the rat renal cortex. These bindings were evaluated to be specific by means of computerized image analysis system (RAS 1,000) of ARG. Micro-autoradiogram showed that the tubules in the renal cortex had higher specific grain density than the glomeruli. These findings suggested that alpha 1-adrenoceptor in the rat kidney located mainly on the cortical tubules.

Adrenergic alpha-Antagonists↗
PubMed1988

Localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus: in vitro electron microscopic autoradiographical investigation using 125I-labeled ANP.

The localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus of the rat was studied by electron microscopic autoradiography using 125I-labeled ANP. A total of 1,134 silver grains counted in 30 glomeruli were distributed as follows: processes of the podocytes (36.4%), cell bodies of the podocytes (14.8%), basement membrane (12.3%), endothelial cells (6.3%), mesangial cells (7.7%) and others, for example the vascular spaces (22.5%). This finding indicates that ANP binding sites are mainly localized on the foot processes of the podocytes in the glomerulus.

Animals↗
PubMed1988

Properties of [3H]bunazosin binding in rat kidney.

Properties of alpha 1-adrenoceptor binding in the renal cortex of rats were studied with [3H]bunazosin as the radiolabeled ligand. Both temperature and incubation time influenced bunazosin receptor binding. Subcellular distribution of [3H]-bunazosin binding revealed that the membrane fraction (30,000 x g pellet) had the largest proportion of total binding sites. Pretreatment of membrane preparation at 56 degrees C for 30 minutes greatly decreased the specific binding of [3H]-bunazosin, though there was no significant effect of the pretreatment at 24 degrees C and 37 degrees C for 30 minutes. Among various cations, some divalent ions such as Cu++ and Zn++ greatly decreased bunazosin binding, whereas monovalent ions had no effect on specific binding. Results of Scatchard analysis suggested that the rat renal cortex membrane has single binding sites with an apparent dissociation constant of 0.38 +/- 0.06 nmol/L, though the rat medulla membrane has no potent binding activity with bunazosin. The displacement study revealed that various adrenergic agents inhibit [3H]bunazosin binding in dose-dependent fashion; the rank order of potencies was bunazosin prazosin > phentolamine >> dl-norepinephrine > clonidine, yohimbine >> pindolol, propranolol. These findings reveal that bunazosin has specific receptor binding in the rat renal cortex, indicating that alpha 1-adrenoceptors exist in the rat renal cortex.

Adrenergic alpha-Antagonists↗
PubMed1987

Differential changes in atrial natriuretic peptide and vasopressin receptor bindings in kidney of spontaneously hypertensive rat.

To elucidate the role of atrial natriuretic peptide (ANP) and vasopressin (VP) in a hypertensive state, ANP and VP receptor bindings in spontaneously hypertensive rat (SHR) kidney were analyzed using the radiolabeled receptor assay (RRA) technique. Systolic blood pressure of SHR aged 12 weeks was statistically higher than that of age-matched Wistar Kyoto (WKY) rats. Maximum binding capacity (Bmax) of [125I]-ANP binding to the SHR kidney membrane preparations was statistically lower than that of WKY rats, but dissociation constant (Kd) was not significantly different. On the other hand, Bmax of [3H]-VP binding to the SHR kidney membrane preparations was statistically higher than that of WKY rats, but Kd were similar. Since the physiological action of ANP is natriuresis and VP is the most important antidiuretic hormone in mammalia, these opposite changes of ANP and VP receptor bindings in SHR kidney suggested that these peptides may play an important role in the pathophysiology of the hypertensive state, although it has not been confirmed as yet.

Animals↗
PubMed1986

Effect of indapamide on atrial natriuretic polypeptide receptor in spontaneously hypertensive rat kidney.

The effect of a hypotensive state on atrial natriuretic polypeptide (ANP) receptors of the kidney treated by indapamide was investigated in spontaneously hypertensive rats (SHRs). SHRs aged 12 weeks were injected intraperitoneally with indapamide (10mg/kg/day) for 10 days and an ANP radiolabeled receptor assay (RRA) was done on the 11th day. The systolic blood pressure of SHRs injected with indapamide (IDP group) was statistically lower than that of SHRs injected with 2% gum Arabic solution (control group). Concerning the RRA of ANP in the SHR kidney, high affinity and low capacity binding sites were observed in the IDP group (Kd = 0.220 +/- 0.059 nM, Bmax = 6.10 +/- 2.36 fmol/100 micrograms) compared with the control group (Kd = 0.401 +/- 0.147 nM, Bmax = 9.96 +/- 2.50 fmol/100 micrograms). These finding suggested that the hypotensive state induced by treatment with indapamide may change the ANP receptor in kidneys of a SHR.

Animals↗
PubMed1985

Clinical and endocrine studies on patients with amenorrhoea associated with weight loss.

Two hundred and forty-three patients with amenorrhoea associated with weight loss were studied. At the onset of amenorrhoea, regardless of percentage weight loss, basal levels of LH were low and LH responses to LHRH were impaired. However, both basal and stimulated levels of FSH were comparable to normal. With resumption of menstruation, the basal and stimulated levels of LH were found to rise to normal, while FSH responses continued to exceed normal. However, 16.6% of 66 unimproved cases had normal responses but remained amenorrhoeic. Furthermore, amenorrhoea persisted in 71% of 31 patients with complete recovery of body weight. No significant correlation was noted between percentage weight loss and responsiveness to LHRH, nor between recovery of body weight and resumption of menstruation. Return to normal weight is desirable for resumption of normal cyclic menstruation and hypothalamic-pituitary function, but is not always effective.

Adolescent↗
PubMed1984

Image analysis of the complex of actin-tropomyosin and myosin subfragment 1.

A three-dimensional image of the "rigor" complex of actin-tropomyosin-S1 was reconstituted from both low dose (10 electrons/A2) and high dose (greater than 500 electrons/A2) electron microscopic images of specimens embedded in unbroken and unbacked stain sheets of uranyl acetate over the holes of perforated carbon films. Myosin S1 shows multi-domain submolecular structure as has been earlier observed in actin-S1 ( Wakabayshi & Toyoshima, 1981) and actin-heavy meromyosin ( Katayama & Wakabayashi , 1981). The morphological unit of the actin-tropomyosin-S1 was found to be composed of at least three domains (domains A, B and D) and three regions (C, E and H). A myosin S1 molecule has a complex shape, which cannot be represented by a simple rod with one major axis. The shape of S1 should be approximated by at least two rods. The domain D is identified as the main part of S1. The angle between the major axis of this domain and the axis of actin helix was about 72 degrees, which is almost right angle. The angle between the axis of actin helix and major axis of the region E, which is less bulky than the domain D and makes no contact with actin, is much smaller than the value for the domain D. The resolution of reconstituted images from both high and low dose micrographs was improved so that the radial resolution became about 15 A and the axial one became about 25 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1984

Proximity of reactive lysyl residue to the antigenic site in rabbit skeletal myosin against the monoclonal antibody (MF-18) generated to chicken skeletal myosin.

MF-18, one of the monoclonal antibodies generated to chicken myosin, cross-reacted with rabbit skeletal myosin subfragment-1 (S1). Utilizing an improved procedure of immuno-blotting, a decrease in reactivity of MF-18 to S1 by trinitrophenylation was observed. This indicates that the reactive lysyl residue is very close to the hapten site. This is consistent with the evidence that the hapten site resides in the 26,000 dalton tryptic fragment of S1. Use of such antibodies as labels may open the way to determining the location of specific hapten sites in the three-dimensional image of actin-S1 complex reconstructed from the electron micrographs.

Animals↗
PubMed1984

[Monolayer culture of human ovarian thecal cells. A study on morphological and functional characteristics].

The aim of this study was to establish a monolayer culture system for human ovarian thecal cells and to investigate their morphological and functional characteristics. Theca layers were isolated and digested with collagenase-hyaluronidase solution, and dispersed thecal cells were cultured for 10 days in plastic dishes. Histological examination indicated that there were no contaminating granulosa cells in isolated theca layers. Various histochemical studies revealed abundant lipid droplets and 3 beta-hydroxysteroid dehydrogenase activity in cultured cells. The major steroids secreted were delta 4-androstenedione (delta 4) and progesterone(P). delta 4 secretion was very high during the first 2 days (31.6 +/- 1.9 ng/1 X 10(5) cells/2 days) and declined thereafter. P was secreted in moderate amounts throughout the 10 day culture period (9.0-21.3 ng/1 X 10(5) cells/2 days), while estradiol secretion was very low. Subsequently, the responsiveness of cultured thecal cells to gonadotropins and dibutyryl cyclic AMP (Bu2cAMP) was investigated. LH/HCG and Bu2cAMP stimulated delta 4 and P secretion in a dose-related manner. The maximal effective doses of LH and HCG were both 10 ng/ml, and that of Bu2cAMP was 10(-3)M. In conclusion, it was evident that these monolayer-cultured human thecal cells could maintain their morpho-functional characteristics during culture. Therefore, this culture system will provide an excellent model for further studies on the functional properties of thecal cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Ca2+-dependent binding of synthetic peptides corresponding to some regions of troponin-I to troponin-C.

We have chemically synthesized two peptides corresponding to the binding regions of troponin-I for troponin-C. Electrophoretic analysis has shown that one of the two peptides binds Ca2+-dependently to troponin-C and the other Ca2+-independently. The biological significance of the results is discussed. The interaction of the peptides with calmodulin was different from that with troponin-C.

Amino Acid Sequence↗
PubMed1981

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. III. The multi-domain structure of actin-heavy meromyosin complex.

Heavy meromyosin (HMM) prepared by chymotryptic digestion retains DTNB light chain and subfragment-2 (S2) in addition to the two subfragment-1 (S1) moieties. The electron micrograph of negatively stained rigor complex of HMM and actin showed some different features as compared with that of S1 and actin. The three-dimensional image reconstructed from the electron micrograph of actin-HMM gave additional domains other than those observed in actin-S1 (Wakabayashi and Toyoshima, 1981). The differences between the images of actin-HMM and actin-S1 were attributed to the differences in protein composition. Further, the structural characteristics of actin-HMM are discussed in comparison with those of the actin-S1.

Actins↗
PubMed1979

Quantitative analysis of the mechanism of negative staining with native collagen fibrils and polar tropomyosin paracrystals.

The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.

Amino Acids↗
PubMed1979

Electron microscopic analysis of tropomyosin paracrystals.

Negatively stained images of divalent cation-induced tropomyosin paracrystals show polymorphic patterns which are almost bipolar. Although these bipolar forms are naturally due to antiparallel arrays of molecules, the precise molecular arrangements have not been clarified yet except in the case of one type of these polymorphic paracrystals by Stewart and McLachlan [(1976) J. Mol. Biol. 103, 251--269]. In the previous paper we showed that the lead-induced polar paracrystal is a parallel and in-register array of tropomyosin molecules. Moreover, we have made it possible to locate a given residue on the staining pattern. By overlapping two photographic transparencies of the polar paracrystal antiparallel, directly observed images of polymorphic bipolar paracrystals could be synthesized photographically with fidelity. The overlap length between N-terminals of antiparallel pairs of molecules could be easily determined without any assumptions. Next, we considered the stabilizing forces involved in the morphogenesis of such polymorphic paracrystals. The cation-bridged attractive forces already proposed by some groups were insufficient to account for the stability of some specific forms of tropomyosin paracrystals. From the primary amino acid sequence of tropomyosin, we calculated the changes of repulsive forces between the basic residues with changes of molecular overlap length between the N-terminals of antiparallel pairs. By setting the values of charge appropriately, we could account well for the stability of the polymorphic structures observed by electron microscopy.

Amino Acid Sequence↗