Biomedical subjects

Y Shimura

Publications and source records attributed to Y Shimura.

At least 37 records · Page 2Linked to original sources

PubMed1997

Restoration of seminiferous tubular function after discontinuation of long-term gonadotropin-releasing hormone agonist administration in premature male rats.

We examined whether seminiferous tubular function develops normally after discontinuation of long-term gonadal suppression in premature male rats. Wistar male rats 4 weeks old were subjected to the injection of gonadotropin-releasing hormone (Gn-RH) agonist or normal saline solution as control for 12 weeks. The rats were sacrificed 0 and 6 weeks after discontinuation of the treatment. Histological examinations of the seminiferous tubules immediately after cessation of the Gn-RH agonist treatment demonstrated a stage-related change in specific germ cells. Seminiferous tubules of Gn-RH agonist-treated rats were narrow and irregular in shape, and contained significantly fewer spermatids and pachytene spermatocytes at stages VII to XIV than those in controls. A complete development of spermatogenesis was histologically observed 6 weeks after cessation of the treatment. Leydig cells became atrophic without any reduction in cell number immediately after the treatment, but Leydig cells grew rapidly and were similar in appearance to those in control rats 6 weeks after cessation of the treatment. Serum testosterone concentrations were noticeably suppressed immediately after cessation of the treatment (P < 0.01 vs. control) and reached a similar level to those of controls 6 weeks after the cessation. Testes weights were significantly lower in Gn-RH agonist-treated rats than in control rats and had not fully developed 6 weeks after cessation of the treatment (P < 0.01 vs. control). These results suggest that the testicular function develops normally after cessation of the long-term gonadal suppression in premature rats, although the increase in testicular weight may be slightly influenced.

Animals↗

PubMed1997

[Lipoprotein(a) concentrations and apolipoprotein(a) phenotype frequencies in cord blood of newborn infants and sera of children in Japan].

Lipoprotein(a) [Lp(a)] is considered to be an additional, independent, and largely genetically risk factor for the development of premature coronary heart disease. We measured Lp(a) levels and apo(a) phenotype in cord blood samples from 140(72 males, 68 females) Japanese newborn infants, and those in serum samples from seventy-nine normolipidemic out-patient children from the pediatrics. In the present study, we determined the relations between cord blood Lp(a) levels and apo(a) phenotypes. Maternal age, gestational age, weight and height were analyzed as covariates. The distribution of Lp(a) levels in cord blood was highly skewed towards the lower levels, as previously described. The mean Lp(a) concentration was 4.6 mg/dl(SD of 2.22 mg/dl) and median of 4.0 mg/dl, approximately three-fold lower than that observed in Japanese adult subjects. We did not find differences in the mean and median cord blood Lp(a) levels between male and female. Phenotyping of apo(a) was performed for 140 sera. The mean Lp(a) level in the B type was almost three times higher than that for the S4 type. A strong inverse relationship was found between the apparent molecular weight of apo(a) isoforms and serum Lp(a) concentration. The concentrations of Lp(a) reached to adult levels within first five years of life.

Adult↗

PubMed1996

A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome.

Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm. As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome. Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA. In budding yeast, RNA helicase-like protein Prp22 is implicated in this process. Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1). dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro. Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus. The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome. This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor.

Amino Acid Sequence↗

PubMed1996

Factors involved in the activation of pre-mRNA splicing from downstream splicing enhancers.

The excision of introns with weak polypyrimidine tracts at their 3' splice sites can be enhanced by sequence elements in the downstream exon or by a downstream 5' splice site. The enhancers inside the exon do not conform to a strict consensus, but they are generally rich in purines. Here, we show that members of the family of SR proteins recognize these elements. Not only does SF2/ASF activate many different polypurine enhancers, but also at least one other SR protein, most likely SC35, is active as well. The degree of splicing activation varies with the polypurine enhancers and the SR proteins. Further, we show that the similar activation by downstream 5' splice sites requires U1 snRNP, which is not the case with purine-rich enhancers. These results are consistent with a model showing that U1 snRNP binds to the 5' splice site and SR proteins to exonic sequences upstream of the 5' splice site. Both interact with U2AF at the 3' splice site. This represents a molecular explanation for the exon recognition which is important for splice site selection in mammals.

Alpharetrovirus↗

PubMed1996

Listener and context dependency in the perception of emotional aspects of infant voice.

Developmental aspects of an infant's ability to express emotions through vocalizations were studied based on perceptual rating experiments against 12 vocalization- and emotion-related reference words. Three groups of listeners, students, mothers with infants, and nursery governesses, rated 28 voice samples recorded from a male infant at 6, 9, 12 and 17 months of age, under a positive or negative context. Among three factors extracted by a factor analysis, one representing the emotional contrast of frightened/angry versus happy was found to be independent of listener group, infant age and context. The other two, one representing contrast between pleased/happy versus demanding/sad and the other seeking affection and rejecting/angry were dependent on infant age and context. These results may indicate that infants even at 6 months of age can express the emotional contrast of 'pleasure' versus 'discomfort' through vocalization, which listeners perceive independently in context.

Anger↗

PubMed1996

A Y-box protein is a suppressor factor that decreases thyrotropin receptor gene expression.

The decanucleotides in a tandem repeat, -162 to -140 bp, are suppressor elements that decrease TSH receptor (TSHR) gene expression by different mechanisms. A factor(s) interacting with the 3'-decanucleotide compete for proteins that bind the cAMP response element, -139 to -132 bp, a constitutive enhancer necessary for efficient TSHR expression. The 5'-decanucleotide is in a CT-rich, S1 nuclease-sensitive region of the promoter; its suppressor activity has been related to its ability to bind a nonthyroid-specific protein to its coding strand. In this report we clone a complementary DNA encoding a single strand DNA-binding protein that forms a specific protein-DNA complex with the coding strand of the 5'- but not the 3'-decanucleotide and not with the 5'-decanucleotide noncoding or double strand. We show, by cotransfection with TSHR promoter-chloramphenicol acetyltransferase chimeras, that the protein is a suppressor that regulates the function of the 5'- but not the 3'-decanucleotide. The protein is a Y-box protein that was previously cloned as an enhancer factor from the rat liver; it is, however, 95% identical to human YB-1, which suppresses major histocompatibility class II gene expression, and to human nuclease-sensitive element protein-1, a Y-box protein identified by its ability to bind single strand, CT-rich, nuclease-sensitive elements of genes that, like the TSHR, have GC-rich promoters. Unexpectedly, the Y-box protein binds two other sites in the minimal TSHR promoter in a single strand-specific fashion and acts a suppressor at each of these sites. One is associated with the insulin response element of the minimal TSHR promoter and is not in an overtly CT-rich region. The other is located 3' to the cAMP response element in a region termed the S-box, -120 to -113 bp, because of its homology to the S-box of the major histocompatibility class II promoter; this site is in a CT-rich area and, as in the class II promoter, is linked to cAMP-induced gene suppression. A conserved CCTC sequence in each site is important for the binding and suppressor function of the Y-box protein.

Amino Acid Sequence↗

PubMed1995

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing alpha-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47.

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Amino Acid Sequence↗

PubMed1995

Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in pre-mRNA splicing and nuclear export of RNA. As a candidate for the factor involved in these nuclear events we have previously purified an 80 kDa nuclear cap binding protein (NCBP) from a HeLa cell nuclear extract and isolated its full-length cDNA. In this report, in order to obtain a clue to the cellular functions of NCBP, we attempted to identify a factor(s) that interacts with NCBP. Using the yeast two-hybrid system we isolated three clones from a HeLa cell cDNA library. We designated the proteins encoded by these clones NIPs (NCBP interacting proteins). NIP1 and NIP2 have an RNP consensus-type RNA binding domain, whereas NIP3 contains a unique domain of Arg-Glu or Lys-Glu dipeptide repeats. We also show that NCBP requires NIP1 for binding to the cap structure. Possible roles of NIPs in cap-dependent nuclear processes are discussed.

Amino Acid Sequence↗

PubMed1995

Developmental and tissue-specific regulation of the gene for the wheat basic/leucine zipper protein HBP-1a(17) in transgenic Arabidopsis plants.

Wheat basic/leucine zipper protein HBP-1a(17) binds in vitro specifically to ACGT motif-containing cis-acting elements, such as the type I element of plant histone promoters and the G-box of hormone- and light-inducible promoters. To address the in vivo function of HBP-1a(17), we isolated and structurally analyzed the HBP-1a(17) gene and examined its expression in transgenic Arabidopsis plants. The HBP-1a(17) gene is composed of 14 exons; the basic region and leucine zipper are encoded by separate small exons, as is the case for other bZIP protein genes. The G-box of the HBP-1a(17) promoter bound specifically to HBP-1a(17) and its related HBP-1a isoforms, suggesting that the HBP-1a(17) gene may be autoregulated, although the binding affinity of these proteins in vitro is very low. In Arabidopsis plants, activation of the HBP-1a(17) promoter was highly restricted to photosynthetically active mesophyll, and guard cells and vascular bundles of vegetative leaves. Etiolation of transgenic plants resulted in inhibition of expression of the HBP-1a(17) promoter. Indeed, the HBP-1a(17) promoter contains several sequence elements homologous to cis-acting elements conserved in light-inducible promoters. It is, therefore, assumed that the HBP-1a(17) gene is light regulated and that HBP-1a(17) is involved in light-responsive gene transcription via the G-box.

Amino Acid Sequence↗

PubMed1995

Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.

Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.

Animals↗

PubMed1995

The thyrotropin receptor.

This chapter has outlined the complex process required for thyroid growth and function. Both events are regulated by TSHR via a multiplicity of signals, with the aid of and requirement for a multiplicity of hormones that regulate the TSHR via receptor cross-talk: insulin, IGF-I, adrenergic receptors, and purinergic receptors. Cross-talk appears to regulate G-protein interactions or activities induced by TSH as well as TSHR gene expression. The TSHR structure and its mechanism of signal transduction is being rapidly unraveled in several laboratories, since the recent cloning of the receptor. In addition, the epitopes for autoantibodies against the receptor that can subvert the normal regulated synthesis and secretion of thyroid hormones, causing hyper- or hypofunction, have been defined. Studies of regulation of the TSHR minimal promotor have uncovered a better understanding of the mechanisms by which TSH regulates both growth and function of the thyroid cell. A key novel component of this phenomenon involves TSH AMP positive and negative regulation of the TSHR. Negative transcriptional regulation is a common feature of MHC class I genes in the thyroid. Subversion of negative regulation or too little negative regulation is suggested to result in autoimmune disease. Methimazole and iodide at autoregulatory levels may be important in reversing this process and returning thyroid function to normal. Their action appears to involve factors that react with the IREs on both the TSHR and the TG promoter. Too much negative regulation, as in the case of ras transformation, results in abnormal growth without function. TTF-1 is implicated as a critical autoregulatory component in both positive and negative regulation of the TSHR and appears to be the link between TSH, the TSHR, TSHR-mediated signals, TG and TPO biosynthesis, and thyroid hormone formation. Differentially regulated expression of the TSHR and TG by cAMP and insulin depend on differences in the specificity of the TTF-1 site, that is, the lack of Pax-8 interactions with the TSHR, and the IRE sites. Single-strand binding proteins will become important in determining how TSHR transcription is controlled mechanistically.

Amino Acid Sequence↗

PubMed1995

Characterization of an up-stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter.

A thyroid transcription factor-1 (TTF-1)-binding element in the rat TSH receptor (TSHR) promoter, between -189 and -175 basepairs (bp), is important for both thyroid-specific expression and thyroid-specific TSH/cAMP autoregulation of the TSHR. The identification of an up-stream TTF-1-binding site and its relationship to the function of the down-stream TTF-1 element are the subjects of this report. Sequence analysis identifies a potential TTF-1 site at -878 bp; deoxyribonuclease-I footprinting shows that the -881 to -866 bp region is protected by recombinant TTF-1 protein and by nuclear extracts from FRTL-5 thyroid cells that contain TTF-1, but not by extracts from nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, which have no TTF-1, or by Pax-8. FRTL-5, but not FRT or BRL cell nuclear extracts, form a specific protein-DNA complex with this region in gel mobility shift analyses; its formation is prevented by TTF-1-binding elements from the thyroglobulin promoter. The upstream TTF-1 site acts as an enhancer when coupled to a heterologous simian virus-40 promoter-chloramphenicol acetyltransferase (CAT) chimera and transfected into FRTL-5 thyroid cells. There is a greater increase, 3-vs. 2-fold (P < 0.05), when TSHR promoter-CAT chimeras, which contain the up-stream TTF-1 element, pTRCAT5'-907 or pTRCAT5'-886, as opposed to those in which it is deleted, pTRCAT5'-907 delta USTTF-1, are transfected into FRTL-5 cells or cotransfected with a TTF-1 expression vector into BRL cells, which have no endogenous TTF-1. The TTF-1-dependent activity of pTRCAT5'-907 delta USTTF-1 is the same as that of the minimal promoter, -220 to -39 bp, containing only the down-stream TTF-1 site in both cells. Transfection of chimeric TSHR-CAT plasmids with the down- and/or up-stream TTF-1 site deleted reveals that the down-stream TTF-1 element functions in the absence of the up-stream element, but function of the up-stream site requires the down-stream TTF-1 element. Like the down-stream TSHR TTF-1-binding site, the up-stream TTF-1 site is different from TTF-1 sites in the thyroglobulin and thyroid peroxidase promoter, in that it does not interact with Pax-8.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene expression.

An element, -186 to -176 base pairs (bp), in the minimal TSH receptor (TSHR) promoter binds thyroid transcription factor-1 (TTF-1) and is important for both constitutive expression and TSH/cAMP-induced negative autoregulation of the TSHR in thyroid cells. An element on the noncoding strand of the TSHR, contiguous with the 5'-end of the TTF-1 element, has single strand binding activity. It is distinct from the TTF-1 site, as evidenced by competition experiments using gel shift assays; but the association of the two elements is not random. Thus, the single strand binding protein (SSBP) element also exists contiguous to the 5'-end of an upstream TTF-1 site, -881 to -866 bp; mutation of two conserved nucleotides in each SSBP element results in the loss of SSBP binding and cross-competition. Transfection experiments indicate that full, constitutive TSHR gene expression in FRTL-5 thyroid cells requires the binding of both SSBPs and TTF-1, since mutation of either element halves thyroid-specific promoter activity, whereas mutation of both decreases promoter activity to values near those of a control vector. Transfection experiments with rat liver cells support their independent activities and show that the SSBP site contributes to TSHR gene expression in non-thyroid tissue. The SSBPs function conjointly with TTF-1 in thyroid-specific, TSH/cAMP-induced negative autoregulation of the TSHR. Thus, TSH or forskolin-treated FRTL-5 cells coordinately decrease TSHR RNA levels and TSHR DNA binding to both the SSBPs and TTF-1; also the maximal TSH/cAMP-induced decrease in gene expression requires both elements. The TSH-induced effect in each case is inhibited by cycloheximide; the TSH-induced decrease in SSBP/DNA complex formation requires the presence of insulin or calf serum, exactly as does TSH-induced down-regulation of TSHR RNA levels. In sum, full, constitutive expression of the TSHR in thyroid cells requires TTF-1 and the SSBPs to bind separate, contiguous elements on the TSHR promoter. TSH/cAMP decreases the binding of each factor to its respective site, thereby decreasing TSHR gene expression. The role of the SSBP and TTF-1 sites in constitutive TSHR expression and in TSH/cAMP-induced negative regulation of the TSHR is, therefore, additive and independent.

Animals↗

PubMed1995

Effects of growth hormone and insulin-like growth factor I on testosterone secretion in premature male rats.

Present study was planned to clarify the effects of GH and insulin-like growth factor I (IGF-I) on testosterone secretion using premature male rats. Forty rats were divided four groups. GH, IGF-I, both of them or normal saline solution as control were subcutaneously administered to the rats of each group for seven days from 3-week to 4-week of age. After the treatment, six of each group were used to human chorionic gonadotropin (hCG) loading and four to Leydig cell preparation. Serum testosterone responses to hCG loading were significantly higher in 4-week-old rats treated with GH and/or IGF-I for 1 week than in control rats. However, the responses were similar among three treated groups (GH, IGF-I and both). After one-week treatment with GH and/or IGF-I, isolated Leydig cells were prepared from testes of 4-week-old rats and testosterone production by the stimulation of hCG was examined. Amounts of testosterone production stimulated by hCG were significantly greater in the treated rats than in control rats. These findings suggest that GH mediated by IGF-I promotes the testicular responsiveness to gonadotropin on testosterone production in premature rats.

Analysis of Variance↗

PubMed1995

Aspects of splice site selection in constitutive and alternative pre-mRNA splicing.

RNA splicing is an indispensable step for expression of many eukaryotic genes. Combinations of 5' and 3' splice sites should be correctly selected in both constitutive and alternative splicing. Recent studies have revealed mechanisms of alternative splicing in some systems, in which specific regulators play vital roles in splice site selection. On the other hand, essential splicing factors such as SR proteins modulate splice site usage of general machinery. Specific regulators and splicing factors such as SR proteins have some common structural features. With these related components, a similar machinery of splice site selection is involved in constitutive and alternative splicing.

Alternative Splicing↗

PubMed1995

Touch-inducible genes for calmodulin and a calmodulin-related protein are located in tandem on a chromosome of Arabidopsis thaliana.

Genes for calmodulin and calmodulin-related proteins in Arabidopsis are up-regulated by a variety of physical stimuli, which include rain, wind and touch [Braam and Davis (1990) Cell 60: 357]. We have isolated five genes for calmodulin (AtCAL1, 2, 3, 5, 6) and one gene for a calmodulin-related protein (AtCAL4) from an Arabidopsis genomic library. Touch stimulus of Arabidopsis plants induces the accumulation of mRNA transcribed from AtCAL4 and AtCAL5, but not from the other isolated genes. The two touch-inducible genes are arrayed in tandem with a short intergenic region of 700 bp but they show different organ-specific patterns of expression.

Arabidopsis↗

PubMed1994

Identification of a novel insulin-responsive element in the rat thyrotropin receptor promoter.

By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.

Animals↗

PubMed1994

Cloning of a complementary DNA encoding an 80 kilodalton nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region.

Amino Acid Sequence↗