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Biomedical subjects

S Takemori

Publications and source records attributed to S Takemori.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

Adrenal Cortex↗
PubMed1996

15-lipoxygenase metabolite(s) of arachidonic acid mediates adrenocorticotropin action in bovine adrenal steroidogenesis.

The acute activation of adrenal glucocorticoid synthesis by ACTH has long been believed to be mediated by cAMP as the major second messenger, although increases in cellular cAMP concentration have not been observed at low concentrations of ACTH. We found that steroidogenesis in bovine adrenal fasciculata-reticularis cells was activated by the addition of arachidonic acid or its 15-lipoxygenase metabolite, 15-hydroperoxyeicosatetraenoic acid. The cellular 15-lipoxygenase pathway was significantly activated by 1 pM ACTH, at which concentration no increase in cellular cAMP synthesis was observed. The 1 pM ACTH-induced stimulation of steroidogenesis was completely suppressed by a lipoxygenase inhibitor, AA-861. The stimulation was independent of the increase in cellular cAMP. These results show that the action of 1 pM ACTH on steroidogenesis may be mediated by the 15-lipoxygenase metabolite(s) as a solo second messenger. The addition of ACTH at concentrations higher than 10 pM increased both the 15-lipoxygenase activity and cellular cAMP synthesis. Under these conditions, the 15-lipoxygenase metabolite(s) and cAMP were shown to mediate the activation of steroidogenesis synergistically. The presence of a dual second messenger system could explain the stimulation of steroidogenesis by ACTH at physiological concentrations.

Animals↗
PubMed1996

Comparison of cellular distribution of LH receptors and steroidogenic enzymes in the porcine ovary.

Previous studies have shown a heterogeneous expression of LH receptors in various structures of the porcine ovary. Specially striking was the existence in the preovulatory follicle of inner layers of theca interna cells devoid of LH receptor and the confinement in the corpus luteum of the LH receptor to the external cellular layers. In the present study, we have compared the steroidogenic capabilities of LH receptor-positive and -negative cells using immunocytochemistry for side-chain cleavage P450, 3 beta-hydroxysteroid-dehydrogenase, 17 alpha-hydroxylase P450 and aromatase P450. We have also examined, using the same methods, the evolution of the various cell types after ovulation and during the development of the corpus luteum. In preovulatory follicles the inner layers of theca cells which were not labelled with anti-LH receptor antibodies appeared to express the steroidogenic enzymes in a way similar to that of the outer LH receptor-positive cell layers. Ovulation per se did not change the distribution of LH receptors (present in the outer luteal cells and in the granulosa) or of steroidogenic enzymes. However, 48 h after follicular rupture there as a marked decrease in overall labelling with anti-LH receptor antibody, and especially a disappearance of immunostaining in the luteal cells of granulosa origin. In the mid-luteal phase (6 days after ovulation), the receptor content seemed to increase in the peripheral luteal cells derived from the theca but the receptor did not reappear in the granulosa-derived luteal cells. Thus the down-regulation of LH receptor appeared to be reversible in the external thecal layers but irreversible in the granulosa cells. Furthermore, the distribution of the various steroidogenic enzymes in the corpora lutea delineated granulosa-derived from theca-derived cells and showed that only the external layers of the latter expressed the LH receptor. These results showed the existence in the preovulatory follicle of two theca interna regions expressing the same steroidogenic enzymes but possibly submitted to a different hormonal control. Furthermore, the cells derived from these two regions as well as the cells of granulosa origin showed a distinct pattern of variation of LH receptivity during the development of the corpus luteum. During these studies we also observed that, in the interstitial tissue, only a minority of cells which derived from remnants of atretic follicles expressed both the LH receptor and the steroidogenic enzymes.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Expression and zonal distribution of CYP2D16 in the guinea pig adrenal cortex: relationship to xenobiotic metabolism.

We recently cloned a CYP2D subfamily member (CYP2D16) from a guinea pig adrenal cDNA library and investigated the expression of CYP2D16 in the guinea pig adrenal cortex and its relationship to adrenal xenobiotic metabolism. A modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed three major bands in the molecular mass range of cytochromes P450 in guinea pig adrenal microsomes. Two of the bands were immunoreactive with anti-CYP17 (54 kDa) or anti-CYP21 (52 kDa) antibody. The third band (50 kDa) was immunoreactive with antibody raised against CYP2D1 and with anti-CYP1A1/1A2 antibody. Microsequencing of the 50-kDa band yielded an amino-terminal sequence of 38 amino acids identical to that deducted from the CYP2D16 cDNA. In addition, Northern blot analyses indicated the CYP1A1 was not expressed in the adrenal gland, suggesting that only CYP2D16 composed the microsomal 50-kDa band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses demonstrated greater expression of CYP2D16 in microsomes from the inner zone (zona reticularis) of the adrenal cortex than from the outer zones, coinciding with the major site of adrenal xenobiotic metabolism. Bufuralol-1'-hydroxylase activity, a marker for CYP2D isozymes, was also greater in inner- than in outer-zone microsomal preparations and was highly correlated with CYP2D16concentrations. Northern blot analysis with a full-length CYP2D16 cDNA as the probe gave strong bands with adrenal inner zone RNA preparations and relatively weak bands with outer zone RNA. CYP2D16 mRNA was also detectable in liver and kidney RNA preparations, but at lower levels than in the adrenal inner zone, and it was not detectable in testes, lung, intestines, or heart. Overall, the results demonstrate that CYP2D16 is expressed at highest levels in the inner zone of the guinea pig adrenal cortex and suggest a major role for this isozyme in adrenal xenobiotic metabolism.

Amino Acid Sequence↗
PubMed1995

Kinetic studies of cytochrome P-45017 alpha, lyase dependent androstenedione formation from progesterone.

The reaction mechanism of androstenedione formation from progesterone was analyzed in a membrane reconstituted system consisting of P-45017 alpha, lyase and NADPH-cytochrome P-450 reductase using a rapid quenching device at 10 degrees C. In these rapid quenching experiments, only the metabolites of [3H]progesterone bound to P-45017 alpha, lyase at the initial stage were detectable during the limited cycles of the P-45017 alpha, lyase reactions (1-120 s). The level of 17 alpha-hydroxy[3H]progesterone increased rapidly in a short period (1-5 s) and then decreased to about half. That of [3H]androstenedione increased gradually from 2 s, which exactly corresponded to the decrease in 17 alpha-hydroxy[3H]progesterone. 17 alpha-Hydroxyprogesterone was conclusively the actual intermediate steroid which did not dissociate from P-45017 alpha, lyase during the successive hydroxylation reaction into androstenedione. A kinetic model can clearly describe the successive reaction catalyzed by P-450 17 alpha, lyase, in which progesterone is converted successively into androstenedione via 17 alpha-hydroxyprogesterone, some of which dissociates from the active site of P-450 17 alpha, lyase and is never metabolized into androstenedione. We analyzed the effects of pH and the amount of NADPH-cytochrome P-450 reductase on the successive reaction and proved that the reaction was regulated by the rate of electron transfer for the conversion of the bound 17 alpha-hydroxyprogesterone to androstenedione. Furthermore, we found that the product dissociation from P-450 17 alpha, lyase is the rate-limiting process in the steady-state metabolism of progesterone by P-450 17 alpha, lyase.

17-alpha-Hydroxyprogesterone↗
PubMed1995

Incorporation of bovine adrenal 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase into phospholipid vesicles.

3 beta-Hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD/I) and cytochrome P-450C21 were co-purified from bovine adrenocortical microsomes by an improved method. The 3 beta-HSD/I was successfully incorporated into liposomal membranes in which the enzyme activity was greatly stabilized. Enzymatic activities and kinetic parameters of the 3 beta-HSD/I proteoliposomes were almost the same as those of the solubilized form.

17-alpha-Hydroxypregnenolone↗
PubMed1995

Cytochrome P-450 transfer from adrenocortical submitochondrial particles to liposome membranes.

The transfer of cytochrome P-450 from bovine adrenocortical submitochondrial particles (smp) to unilamellar liposome membranes was investigated using a table top ultracentrifuge. Submitochondrial particles were incubated with liposome membranes at 25 degrees C and precipitated by ultracentrifugation at 200000g for a few minutes at 25 degrees C. All liposome vesicles were recovered in the supernatant. Almost no proteins were detected in the supernatant when only smp were incubated and centrifuged. SDS-PAGE revealed one main protein band for the supernatant when smp were incubated with liposome vesicles at 25 degrees C. This band was reactive to anti-P-450scc IgG. Inaccuracy in time for kinetic studies of the transfer was less than 0.5 min. Transfer of P-450scc from smp to liposome membranes was further demonstrated by the decrease in side-chain cleavage activity of smp for endogenous cholesterol after incubation. Cytochromes P-450 accounted for about 70% of the transferred proteins in the liposome membranes, the amount of which increased exponentially with the incubation time. The inverse value of the relaxation time of the transfer increased linearly with the smp concentration and decreased hyperbolically with the liposome concentration. These results coincide with a mechanism by which cytochrome P-450 dissociates from smp membranes, diffuses, and binds to the liposome membranes. In the transfer of cytochrome P-450, the dissociation from smp membranes was deduced to be the rate-limiting step.

Adrenal Cortex↗
PubMed1995

Structural changes in myosin cross-bridges during shortening of frog skeletal muscle.

X-ray diffraction patterns from frog sartorius muscle were recorded during steady shortening with various loads. The intensity of the third meridional reflection from the thick filament decreased on shortening to an extent proportional to the drop in tension. The intensity correlated more closely with the tension than with the shortening velocity. The Bragg spacing of the third meridional reflection decreased in proportion to the decrease in tension. The intensity decrease of the actin layer lines at 1/5.1 and 1/5.9 nm-1 was roughly proportional to the decrease in the load, indicating that the number of cross-bridges decreases similarly. The intensity of the (1,1) equatorial reflection showed a significant decrease only with low loads. Assuming that a steady structural state is attained during steady shortening, the results are consistent with the cross-bridge model in which the number of myosin cross-bridges decreases during shortening.

Actins↗
PubMed1995

Effects of adenosine diphosphate on the structure of myosin cross-bridges: an X-ray diffraction study on a single skinned frog muscle fibre.

Using a technique to obtain a detailed X-ray diffraction pattern from a single skinned frog muscle fibre, we studied the effects of ADP on the structure and arrangement of myosin heads. An imaging plate and a cooled-CCD X-ray detector were used to record the diffraction patterns. Addition of 1 mM ADP to a rigor fibre increased the intensity of the third-order meridional reflection of the myosin repeat by 50-85%. The intensity of the sixth-order meridional reflection also increased. After removing the ADP, these intensities decreased but did not return to the level before the ADP was added. No significant changes were observed in the intensities of the equatorial reflections and the actin layer-lines. These results suggest that, upon ADP binding, the conformation of a myosin head changes without detaching from actin. The structural change may involve a relative motion between domains of the myosin head by the closure of the cleft to which an ADP molecule binds.

Adenosine Diphosphate↗
PubMed1995

An X-ray diffraction study on a single frog skinned muscle fiber in the presence of vanadate.

Using a technique to obtain a detailed X-ray diffraction pattern from a single frog skinned muscle fiber with synchrotron radiation and an imaging plate, we studied the arrangement of myosin heads to which ADP and vanadate are bound. The presence of 1 mM vanadate during contraction caused trapping of ADP and vanadate on the myosin head. Both in the presence and absence of Ca2+, the intensities of the equatorial reflections indicated that most of the heads with ADP and vanadate were located close to the backbone of the thick filament. The presence of the first myosin layer-line at 43 nm-1 also suggested that the heads formed a helix around the shaft of the thick filament. Weak intensity of actin layer-lines suggested that the myosin heads were detached from the thin filament. The results suggest that the myosin-ADP-vanadate complex has a weak affinity toward actin regardless of the state of the regulatory system on the thin filament.

Adenosine Diphosphate↗
PubMed1995

Possibility of thermosensitive magnetoliposomes as a new agent for electromagnetic induced hyperthermia.

This study examined a possibility of dextran magnetite (DM)-incorporated thermosensitive liposomes, namely thermosensitive magnetoliposomes (TMs), as a new hyperthermic agent. The temperatures of TM suspensions and cancer tumors injected with TM suspensions were efficiently elevated up to 42 degrees C by electromagnetic induced heating at a frequency of 500 kHz under both in vitro and in vivo conditions. Thus, a possibility of TMs for selective hyperthermia was demonstrated. The temperature rises obtained at various concentrations of TMs suggest that approximately 15 mg Fe/cm3 tumor volume is adequate as a therapeutic dose of TMs for efficient selective hyperthermia.

Animals↗
PubMed1994

Inhibition of adrenal cytochromes P450 by 1-aminobenzotriazole in vitro. Selectivity for xenobiotic metabolism.

Studies were done to determine the effects of a P450 suicide inhibitor, 1-aminobenzotriazole (ABT), on adrenal steroid and xenobiotic metabolism. Incubation of guinea pig adrenal microsomes with ABT plus an NADPH-generating system caused a time-dependent decline in total P450 concentrations. The maximal decrease in P450 levels was approximately 35% and was accompanied by an equimolar decrease in heme content. Western blot analyses indicated that ABT had no effect on P450 apoprotein levels. Benzphetamine (BZ) N-demethylase and benzo[a]pyrene (BP) hydroxylase activities were inhibited almost completely by microsomal incubations with ABT. In contrast, neither steroid 17 alpha-hydroxylase nor 21-hydroxylase activity was affected by ABT. The steroid-induced type I spectral change in adrenal microsomes also was not affected by ABT, whereas that induced by BZ was eliminated. Similar studies with adrenal mitochondria indicated that ABT had no effect on mitochondrial P450 concentrations or on mitochondrial steroid metabolism. The results demonstrate that the in vitro actions of ABT on adrenal cytochromes P450 are highly selective for those isozymes that catalyze xenobiotic metabolism. Therefore, ABT should serve as a useful probe for further characterization of adrenal xenobiotic-metabolizing P450 isozymes.

Adrenal Glands↗
PubMed1994

Regulation mechanism of the catalytic activity of bovine adrenal cytochrome P-450(11)beta.

In our previous paper (Ikushiro et al. (1992) J. Biol. Chem. 267, 1464), two catalytic states were proposed for bovine adrenocortical P-450(11)beta at 37 degrees C: one in liposome membranes and the other in liposome membranes containing P-450scc. Similar reaction characteristics were observed at 5 degrees C and all the experiments in this study were performed at 5 degrees C. P-450(11)beta-proteoliposomes had relatively low 11 beta-hydroxylase activity and could catalyze aldosterone formation from 11-deoxycorticosterone. Relatively high 11 beta-hydroxylase activity was observed in P450(11)beta-proteoliposomes containing P-450scc and in Tween-20 solubilized P-450(11)beta, in which no aldosterone formation could be detected. Optical titration indicated binding of corticosterone to P-450(11)beta to be much weaker in the Tween-20 solubilized state than in proteoliposomes. Corticosterone competitively inhibited 11 beta-hydroxylation reaction of P-450(11)beta-proteoliposomes, but neither in P-450(11)beta-proteoliposomes containing P-450scc nor in the Tween-20 solubilized system. The binding of corticosterone to P-450(11)beta was concluded quite weak in proteoliposomes in the presence of P-450scc and in the Tween-20 solubilized state. Aldosterone formation thus was not possible in these systems. Inability of the bovine adrenocortical zonae fasciculata and reticularis to produce aldosterone may be due to the weak binding of corticosterone to P-450(11)beta in these zones.

Adrenal Cortex↗
PubMed1994

Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase.

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Membrane topology of bovine adrenocortical cytochrome P-450C21: structural studies by trypsin digestion in vesicle membranes.

Purified adrenocortical microsomal P-450C21 was incorporated into vesicle membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. Trypsinolysis of the incorporated P-450C21 resulted in the formation of 30-, 25-, and 20-kDa fragments. Similar fragment formation was observed by trypsinolysis of bovine adrenocortical microsomes with Western blotting using anti-P-450C21 IgG. In the detergent-solubilized state, trypsin cleaved P-450C21 into very small peptides. Washing of the trypsin-treated vesicles with 500 mM Na2CO3 failed to cause these fragments to separate from membranes. N-Terminal amino acid sequencing of these fragments showed that trypsin cleaved the 267 Arg-268Val and 332Arg-333Val bonds of P-450C21. The time course of fragment formation indicated that trypsin cleaved the 267Arg-268Val bond first to produce 30- and 25-kDa fragments and subsequently the 332Arg-333Val bond in the 25-kDa fragment to produce the 20-kDa fragment. Neither 21-hydroxylase activity, the reduced CO difference spectrum, nor the EPR spectrum of digested P-450C21 differed from those of undigested P-450C21. Heat treatment at 50 degrees C for 20 min did not cause any decrease in activity of digested P-450C21, when the substrate progesterone was present. This high stability toward heat treatment was not observed in the solubilized state. Rotational diffusion experiments on P-450C21 showed that the size of the molecule holding the heme was not changed significantly after digestion. On the basis of these results, P-450C21 is concluded to be deeply embedded in the vesicle membranes.

Adrenal Cortex↗
PubMed1993

An X-ray diffraction study of frog skeletal muscle during shortening near the maximum velocity.

Using an imaging plate exchanger and a synchrotron X-ray source, X-ray diffraction patterns were recorded from frog skeletal muscles shortening near the maximum velocity from a sarcomere length of 2.6 to 2.1 microns. The major findings were as follows: (1) the intensity of the second-order myosin meridional reflection, which decreased to 18% of the resting value during isometric contraction, increased to 34% during shortening, showing that the perturbation in the axial arrangement of myosin heads recovered. (2) The intensity of the third-order myosin meridional reflection increased by 32% on isometric contraction and decreased to 51% of the resting intensity during rapid shortening, showing disorder in the axial rearrangement of the myosin heads. (3) The axial Bragg spacing of the third-order meridional reflection, which increased by 1.22% during isometric contraction, was still 0.47% larger than the resting value during shortening, indicating that the increase is not entirely due to extension of the filament by tension. (4) The intensity ratio of the (1,0) and (1,1) equatorial reflections increased a small extent (from 0.57 to 0.62) during shortening, while the 5.9 nm and 5.1 nm actin layer-lines showed large intensity decreases (126 to 101% and 190 to 124% of the resting intensity, respectively), suggesting that considerable numbers of myosin heads are in the vicinity of the thin filament but not tightly bound to actin molecules during rapid shortening. These results are consistent with a model of muscle contraction in which myosin heads attach to actin in two different manners with weak and strong affinities and show that, during shortening, the number of tightly attached heads decreases by half, while the total number of attached heads decreases only slightly.

Animals↗
PubMed1993

Current X-ray diffraction experiments using a synchrotron radiation source.

A Fuji imaging plate and synchrotron radiation are the most distinct innovations of the last twenty years in the X-ray diffraction experiments on biological materials. Here we present results of recent experiments on skeletal muscles made at Photon Factory, Tsukuba. It is now possible to record a two-dimensional X-ray diffraction pattern from a rabbit or frog single skinned fiber with a 30-sec exposure. Although weaker compared with those from whole muscles, it shows layer-lines up to 5.1 nm. When the fiber is activated by Ca2+, the pattern changes in a way similar to that observed when a live muscle is electrically stimulated. Use of single fibers makes various types of structural experiments much easier than using whole muscles or fiber bundles. Not only suitable for physiological experiments, better diffusion makes it also suitable for biochemical experiments using various kinds of labels. Time-resolved experiments with imaging plates are possible by using an imaging-plate exchanger devised by Dr. Y. Amemiya. By combining this and a fast-acting mechanical shutter, it is possible to record a two-dimensional diffraction pattern from a frog whole muscle shortening at the maximum speed. The pattern thus obtained shows weakening of the 5.1 and 5.9-nm actin layer-lines and the third (14.3 nm) and the sixth (7.2 nm) myosin meridional reflections, compared with the pattern from isometrically contracting muscles. On the other hand, the second meridional reflection from the thick filament is intensified. These results suggest very different arrangement of myosin heads during active shortening from that during isometric contraction.

Animals↗