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A Sakurai

Publications and source records attributed to A Sakurai.

At least 163 records · Page 9Linked to original sources
PubMed1990

The roles of three forms of human thyroid hormone receptor in gene regulation.

We have expressed three forms of human thyroid hormone receptor (hTR alpha 1, alpha 2, and beta) in cultured cells by transient transfection. hTR alpha 1 and beta transfected cells showed increased triiodothyronine (T3) binding capacity, but hTR alpha 2 transfected cells did not. When hTR alpha 1 or beta was cotransfected with pUrGH(S), in which a portion of the rat GH 5' flanking region (-236/-147) was ligated into the CAT reporter plasmid (pUTKAT1), T3 increased CAT gene expression. When hTR alpha 2 was cotransfected with pUrGH(S), T3 did not alter CAT gene expression. When hTR alpha 1 or beta was cotransfected with pUrGH(O), in which a synthetic oligonucleotide representing the TRE from the rat GH 5' flanking region (-189/-160) was substituted for the natural enhancer in pUTKAT1, T3 increased CAT gene expression. When hTR alpha 1 and beta were cotransfected with pUrGH(O), induction by T3 was increased. When hTR alpha 2 was cotransfected with hTR alpha 1 or beta, induction by T3 was decreased. These results indicate that hTR alpha 1 and beta function as native TR, that hTR alpha 1 and beta can recognize the same TRE, that hTR alpha 1 and beta can function additively, and that hTR alpha 2 can inhibit the action of hTR alpha 1 and beta.

Animals↗
PubMed1990

Structural analysis of human thyroid hormone receptor beta gene.

Thyroid hormone receptors (TR) are ligand-dependent, DNA-binding, trans-acting transcriptional factors belonging to the erbA-related steroid/thyroid hormone receptor superfamily. To better understand the structural and functional characteristics of TRs, we isolated the gene encoding human TR beta 1 (hTR beta 1). The coding region of hTR beta 1 is split into at least eight exons. Each exon well correlates with functional domains of hTR beta 1 protein, and the exon/intron organization is highly conserved when compared with the chicken c-erbA gene which encodes an alpha-type chicken TR. We demonstrate that hTR beta has at least two mRNA forms having different lengths of the 3' untranslated region. We also note several nucleotide corrections of hTR beta 1 cDNA sequence.

Animals↗
PubMed1990

A synthetic peptide substrate for selective assay of protein kinase C.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.

Amino Acid Sequence↗
PubMed1990

Preventive effect of several drugs against Pseudomonas aeruginosa infection and the toxicity of combined tumor necrosis factor with lipopolysaccharide: relationship between lethality and the arachidonic cascade.

The participation of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) in Pseudomonas aeruginosa (Pa) infection was examined. The lethal challenge of Pa or TNF and LPS injection could be prevented by pretreatment with anti-TNF antibody, polymyxin B, ONO 1078, or Shosaiko-to. The combined effects of TNF and LPS may be deeply related to the lethality of Pa infection. The activities of leukotriene(LT) C4/D4/E4 or platelet activating factor (PAF) were also related to the lethality of Pa infection, probably due to the subsequently produced TNF which acts in combination with LPS. Activating the host defence mechanism with biological response modifiers like Chinese medicines was effective against Pa infection. One mechanism could involve an activity as an LT inhibitor or PAF antagonist. Following the administration of TNF and/or LPS, the serum levels of arachidonic cascade products underwent various changes. With a combination of TNF and LPS, there was a synergistic increment of prostaglandins, thromboxane, and LT. Following pretreatment with Shosaiko-to, suppression of LTs was dominant even with the combination of TNF and LPS, which might be related to the lethality of the infection or combined TNF with LPS.

Aminoglycosides↗
PubMed1990

Successful use of vancomycin hydrochloride in the treatment of lactulose resistant chronic hepatic encephalopathy.

Vancomycin hydrochloride (2 g daily) was administered to 12 patients with cirrhosis and lactulose resistant portal systemic encephalopathy in a double blind crossover trial. All 12 patients showed a remarkable clinical improvement after vancomycin treatment. The mean (SE) electroencephalographic (EEG) frequency changed from 6.3 (0.2) to 8.5 (0.2) cps (p less than 0.001) and the mean arterial ammonia concentration from 152 (4) micrograms/ml to 97 (8) micrograms/ml (p less than 0.001). Their clinical condition deteriorated when treatment was switched to lactulose, returning to the previous slower EEG frequency and high arterial ammonia concentrations. Vancomycin seems to be effective in chronic portal systemic encephalopathy in patients who are not helped by lactulose alone.

Ammonia↗
PubMed1990

Characterization of site-specific polyclonal antibodies to c-erbA peptides recognizing human thyroid hormone receptors alpha 1, alpha 2, and beta and native 3,5,3'-triiodothyronine receptor, and study of tissue distribution of the antigen.

The translated products of v-erbA-related cDNAs have been demonstrated to be thyroid hormone receptors, and three different forms of receptor (alpha 1, alpha 2, and beta) have been found in human tissues. We synthesized five peptides corresponding to different portions of these three receptors and raised site-specific polyclonal-antipeptide sera in rabbits. Each antibody displayed high titer and specificity for its respective antigen when tested in an enzyme-linked immunosorbent assay. Each immunoprecipitated the corresponding in vitro translated products of human c-erbA alpha 1, alpha 2, or beta. Two of the antisera were specific for beta, one for alpha 2, and one detected a sequence common to alpha 1 and alpha 2. The fifth was directed toward the DNA-binding area of the proteins and interacted with each receptor. The four antibodies against alpha 1 and beta immunoprecipitated the native thyroid hormone receptor from rat liver and caused a partial shift in the elution profile of the native receptor labeled with [125I]T3 on Sephacryl S-300 column chromatography. The antibody against alpha 2 protein did not interact with native thyroid hormone receptor from rat liver. Using the indirect immunofluorescence technique with the five antibodies, we detected immunoreactivity primarily in the nucleus of cells in several tissues. In general, there was coordinate expression of both alpha and beta receptors in each organ examined, in agreement with previous data on tissue distribution of mRNAs for human thyroid hormone receptors. These studies prove the identity of v-erbA-related gene products with native thyroid hormone receptors and the expression of both alpha and beta receptors in nuclei of human and rat tissues.

Animals↗
PubMed1990

A human early response gene homologous to murine nur77 and rat NGFI-B, and related to the nuclear receptor superfamily.

When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor alpha 2 cDNA at low stringency, we found a weakly hybridizing cDNA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it.

Amino Acid Sequence↗
PubMed1990

Dominant negative transcriptional regulation by a mutant thyroid hormone receptor-beta in a family with generalized resistance to thyroid hormone.

An abnormal human thyroid hormone beta-receptor (hTR beta-Mf), which has a glycine to arginine substitution in the hormone-binding domain, has been identified in affected members of one family with generalized resistance to thyroid hormone. To better understand the mechanism by which this mutation produces the observed abnormality, expression vectors for the wild-type and mutant thyroid hormone receptors (TRs) were prepared to test hormone-binding activity and trans-activation function. Nuclear extracts of COS-7 cells transfected with wild-type TRs showed specific T3-binding activity, while mutant receptor-transfected COS-7 nuclear extract failed to bind T3. On the other hand, in a avidin-biotin complex DNA-binding assay, in vitro translated hTR beta-Mf showed high binding activity to the thyroid hormone response element, which was indistinguishable from that of wild-type TRs. In a transient expression study, only the wild-type TRs activated a rat GH gene promoter-chloramphenicol acetyltransferase fusion gene in a T3-dependent manner. Additionally, when wild-type TR and hTR beta-Mf were cotransfected, hTR beta-Mf inhibited gene activation regulated by wild-type TRs. From these results we conclude that 1) hTR beta-Mf has no demonstrable T3 binding and appears to have minimal, if any, ability to activate a thyroid hormone-responsive gene in spite of its preserved ability to bind to a TRE in DNA; 2) hTR beta-Mf inhibits the transcriptional activation of a thyroid hormone-responsive gene by the wild-type TRs in a dominant manner; and 3) the dominant negative regulatory function of hTR beta-Mf appears to explain the clinical manifestations of thyroid hormone resistance produced by this mutation when present in the heterozygous state.

Animals↗
PubMed1989

Purification and characterization of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine binding protein in rat kidney.

The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein (CTBP) has been purified over 30,000-fold from rat kidney by using charcoal extraction, Mono Q-Sepharose, Blue Sepharose CL-6B, and Sephacryl S-200 column chromatography. Purified CTBP had a sedimentation coefficient of 4.7 S, Stokes radius of 32.5A, and calculated molecular weight of 58,000. The apparently homogeneous protein consisted of a single polypeptide chain with Mr of 58,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Scatchard analysis of T3 binding showed that NADPH increases maximal binding capacity without changes in the affinity constant (Ka = 2.43 X 10(9) M-1). Double reciprocal analysis of NADPH and binding capacity gave maximal binding capacity of 16,400 pmol/mg of CTBP, Mr = 58,000. The order of affinity of iodothyronine analogues to purified CTBP was as follows: L-T3 = D-T3 greater than triiodothyroacetic acid greater than L-thyroxine. [125I]T3 bound to purified CTBP spontaneously dissociated from CTBP at 20 degrees C (t 1/2 = 22 min) in the absence of NADPH, whereas the dissociation was not observed in the presence of NADPH. The optimal pH for T3 binding was 7.2-7.5 Na+, K+, Ca2+, and Mg2+ (0-200 mM) did not influence T3 binding to CTBP. The purified CTBP did not bind to DNA and was not adsorbed to concanavalin A-Sepharose.

Animals↗
PubMed1989

Evidence for the presence of two active forms of cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) in rat kidney. Specialized functions of two CTBPs in intracellular T3 translocation.

Cytosolic NADPH-dependent 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) purified from rat kidney was further characterized in its T3 binding and its interaction with nuclei. Pretreatment of the CTBP with NADP induced dithiothreitol (DTT)-dependent T3 binding. The DTT-dependent T3 binding was increased by NADP in a concentration-dependent manner, and the maximal binding was obtained by 0.1 microM NADP. Higher concentrations of NADP (more than 0.1 microM), however, reduced T3 binding. NAD also induced DTT-dependent T3 binding, but was very low compared to that induced by NADP. NADPH and NADH did not produce DTT-dependent T3 binding. This NADP-activated, DTT-dependent T3 binding was characterized as follows: Ka for T3 binding was 1.8 x 10(9) M-1, and the maximal binding capacity was 15,000 pmol/mg of protein in the CTBP activated by 0.1 microM NADP. The molecular weight of the CTBP was 58,000 (4.7 S). A complex of [125I]T3 and CTBP (NADP.DTT.CTBP.[125I]T3), which was made from the CTBP pretreated with NADP and DTT, did not bind to DNA. However, the complex bound to the nuclei prepared from rat kidney. Treatment of the nuclei with 0.38 M KCl and with DNase I did not lead to loss of the binding activity for the complex. Treatment of nuclei with 0.5 M NaCl led to the loss of the activity for binding the complex. A complex of [125I]T3 and NADPH-activated CTBP did not bind these nuclear preparations. These results suggested that the active form of CTBP is present in two different forms: one is NADPH-activated, which plays a role as a reservoir for cytoplasmic T3, and the other is NADP-activated, which plays a role as a T3 carrier protein that transfers T3 from cytoplasm to nucleus.

Animals↗
PubMed1989

Differences in nuclear thyroid hormone receptors among species.

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3'-triiodo-L-thyronine (T3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 +/- 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T3 at 22 degrees for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 +/- 1.47 x 10(5) M-1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 +/- 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T3-receptor complex using trypsin alpha-chymotrypsin, elastase, and papain produced distinct T3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.

Animals↗
PubMed1989

Experimental studies on complete global brain ischemia using the isohistogenic infantile head transplant model in Lewis rats.

A new experimental model is presented, using isohistogenic Lewis rats and microsurgical techniques. Transplantation of the head and forelimbs of the infantile rat was done to the groin of an adult rat. The goal was to study the effects of ischemia on brain tissue. This model also proved useful for other experiments in studies of facial and cranial bone growth. The model presented proved efficient for the study goals, and long-term experiments were possible. The maximum ischemia time tolerated by brain tissue was found to be somewhere between 60 min and 120 min.

Animals↗
PubMed1989

Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor beta.

The syndrome of generalized resistance to thyroid hormone is characterized by elevated circulating levels of thyroid hormone in the presence of an overall eumetabolic state and failure to respond normally to triiodothyronine. We have evaluated a family with inherited generalized resistance to thyroid hormone for abnormalities in the thyroid hormone nuclear receptors. A single guanine----cytosine replacement in the codon for amino acid 340 resulted in a glycine----arginine substitution in the hormone-binding domain of one of two alleles of the patient's thyroid hormone nuclear receptor beta gene. In vitro translation products of this mutant human thyroid hormone nuclear receptor beta gene did not bind triiodothyronine. Thus, generalized resistance to thyroid hormone can result from expression of an abnormal thyroid hormone nuclear receptor molecule.

Alleles↗
PubMed1989

Counterregulation of nuclear 3,5,3'-triiodo-L-thyronine (T3) binding by oxidized and reduced-nicotinamide adenine dinucleotide phosphates in the presence of cytosolic T3-binding protein in vitro.

The role of cytosolic T3-binding protein (CTBP) in the regulation of nuclear T3 binding was studied in vitro. Nuclear [125I]T3 binding was observed in the presence of 1.0 mM dithiothreitol (DTT). When the nuclei prepared from rat kidney were incubated with inactive form of CTBP which was also prepared from rat kidney, [125I]T3 binding to nuclei was not affected. When the nuclei were incubated with inactive form of CTBP in the presence of NADP, [125I]T3 binding to nuclei was increased, whereas binding was diminished when nuclei were incubated with CTBP in the presence of NADPH. The inactive form of CTBP was activated by NADPH. NADP also activated CTBP in the presence of DTT. Both active forms of CTBP were again inactivated by extraction with charcoal, and these inactive forms were reactivated by NADPH or by NADP and DTT, but not by NADP alone. Although the nuclei treated with 0.3 M NaCl lost the binding activity for [125I]T3 in the absence of NADP, the nuclei retained the binding activity for [125I]T3 in the presence of NADP and the inactive form of CTBP. Treatment of the nuclei with 0.5 M NaCl lost the binding activity for [125I]T3 not only in the absence but also in the presence of NADP and CTBP. These results suggested that NADP and NADPH play roles as counterregulatory factors for nuclear T3 binding in the presence of CTBP. Further, it was speculated that binding sites for the T3-CTBP complex, which is generated in the presence of NADP and DTT, are present in nuclei, and that binding sites for the complex are different from nuclear T3 receptors.

Animals↗
PubMed1989

Expression of three forms of thyroid hormone receptor in human tissues.

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.

Blotting, Northern↗
PubMed1989

Possible role of histones in the organization of rat liver thyroid hormone receptors in chromatin.

The effects of histone subfractions on rat liver thyroid hormone receptor-DNA interaction were examined using an in-vitro DNA-cellulose binding assay. H1 histones bound to DNA showed reversible and potent inhibition of receptor-DNA binding without affecting receptor hormone binding. Poly-lysine, bovine serum albumin, ovalbumin and cytochrome c did not alter receptor-DNA binding. H1 histone subfractions (calf thymus lysine-rich histone (CTL)-1, CTL-2 and CTL-3) showed potent inhibition of receptor-DNA binding indistinguishable from each other. The quantity of H1 histone subfractions bound to DNA was the same. Although each subfraction has different functional properties, inhibition of receptor-DNA binding was a common feature of all the H1 histone subfractions, which is important for the non-random distribution of the receptor in chromatin. Binding of the receptor to core histones was investigated; it was found to bind to core histones more potently than to other proteins (H1 histone, ovalbumin and cytochrome c). Among core histone subfractions, H4 histone bound to the receptor most potently and is the candidate to be one of the acceptor sites of the receptor in chromatin.

Animals↗