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S Shimasaki

Publications and source records attributed to S Shimasaki.

At least 73 records · Page 4Linked to original sources
PubMed1991

Conserved sequence motifs in the small subunit of human general transcription factor TFIIE.

A general initiation factor, TFIIE, is essential for transcription initiation by RNA polymerase II in conjunction with other general factors. TFIIE is a heterotetramer containing two subunits of relative molecular mass 57,000 (TFIIE-alpha) and two of 34,000 (TFIIE-beta). TFIIE-beta is required in conjunction with TFIIE-alpha for transcription initiation. Here we report the cloning and expression of a complementary DNA encoding a functional human TFIIE-beta. Recombinant TFIIE-beta could replace the natural TFIIE-beta for transcription in conjunction with TFIIE-alpha. Amino-acid sequence comparisons reveal regions with sequence similarities to: subregion 3 of bacterial sigma factors; a region of RAP30 (the small subunit of TFIIF) with sequence similarity to a sigma-factor subregion implicated in binding to RNA polymerase; and a portion of the basic region-helix-loop-helix motif found in several enhancer-binding proteins. These potential homologies have implications for the role of TFIIE in preinitiation complex assembly and function.

Amino Acid Sequence↗
PubMed1991

Site-specific mutagenesis of human follistatin.

Follistatin is a monomeric protein originally discovered in ovarian follicular fluid as a suppressor of pituitary follicle-stimulating hormone (FSH) secretion, and later identified as a binding protein for activin. To explore the role of the Asn-linked carbohydrate chains on the follistatin molecule in regard to the inhibition of FSH secretion and activin binding ability, site-specific mutations were introduced at either or both of the two potential Asn-linked glycosylation sites of human follistatin with 315 amino acids (hFS-315). The three types of follistatin mutants were expressed individually in Chinese hamster ovary cells. When tested for their ability to inhibit FSH secretion and to bind activin, each mutant was found to have a similar property as the non-mutated recombinant hFS-315, suggesting that glycosylation of the follistatin molecule has no effect in these functions. However, a two amino acid insertion in between the second and the third amino acid residues in hFS-315 caused the resulting compound to lose completely its inhibitory activity on FSH secretion from the pituitary as well as its binding ability to activin. This finding suggests that the amino-terminal region of the follistatin molecule is critical for both of these functions.

Activins↗
PubMed1991

Identification of five different insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human.

Five different insulin-like growth factor binding proteins (IGFBPs) were isolated from adult rat serum using gel filtration, ligand affinity chromatography, and two steps of reversed-phase high performance liquid chromatography. Three of them were identified as IGFBP-2, -3, and -4 by their amino-terminal amino acid sequences. One of the remaining two proteins was the rat homologue of the partially characterized IGFBP isolated originally from human cerebrospinal fluid, while the other appeared to be a novel member of the IGFBP family. IGFBP-1 was not found in the adult rat serum under our experimental procedures. cDNAs encoding the novel IGFBP were isolated and characterized from a rat ovary and a human placenta library. The mature protein predicted for both species contained 252 amino acids including 18 cysteines that were located in the homologous positions as IGFBP-1, -2, -3, and -4. We propose to name this protein IGFBP-5. Northern analysis of IGFBP-5 mRNA in rat tissues demonstrated that transcription of this gene is highly active in kidney, although the mRNA was detectable in all tissues examined. Alignment of the amino acid sequences of the five rat IGFBPs revealed a 47-60% similarity, indicating that their individual genes diverged from a single ancestral gene by successive gene duplication in a short time frame during evolution. The chromosomal localizations of IGFBP-1, -2, -3, -4, and -5 genes in human have been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that they were located on chromosomes 7, 2, 7, 17, and 5, respectively.

Amino Acid Sequence↗
PubMed1991

Expression of nicotinic acetylcholine receptor subtypes in brain and retina.

Neuronal nicotinic acetylcholine receptors (AChRs) are composed of two types of subunits: ACh-binding (termed alpha 2, alpha 3, alpha 4 ...) and structural (termed beta 2, beta 3, beta 4 ...). AChR subtypes composed of combinations of subunits of these two types encoded by several related genes are expressed in different parts of the nervous system, where they presumably serve different functional roles. Here we identify the ACh-binding subunit of the most prominent chicken brain AChR subtype by N-terminal amino acid sequence and show that it corresponds to the alpha 4 gene. Previously we identified the structural subunit for this AChR subtype from chicken brain as beta 2 by N-terminal amino acid sequence. Thus, this identifies both genes which encode subunits of the major nicotinic AChR subtype in avian brains. By immunoprecipitation, immunohistochemistry, and northern blot analysis we show that alpha 3 (or a very closely related sequence) is expressed at low levels in the brain and relatively high levels in the retina, while alpha 4 is expressed at high levels in the brain and lower levels in the retina. This differential expression indicates that alpha 3-containing 'ganglionic-type' AChRs may be an important AChR subtype in avian retina.

Amino Acid Sequence↗
PubMed1991

Stage-specific cellular regulation of inhibin alpha-subunit mRNA expression in the rat seminiferous epithelium.

To find out the local regulation of inhibin production and its possible paracrine role in the seminiferous epithelium, inhibin alpha mRNA levels were measured in sequential 1 mm segments of rat seminiferous tubules accurately staged by transillumination technique. Highest levels were found at stages XIV-I-IV of the cycle, and lowest at stages VI-VIIb of the cycle. When dividing spermatogonia were selectively destroyed by 3 Gy of high-energy X-irradiation, stage-specific inhibin alpha mRNA levels remained unchanged until 26 and 38 days after irradiation when stages VII and VIII of the cycle showed 6- and 4-fold increases during a selective reduction of pachytene spermatocyte and round spermatid numbers, respectively. The results suggest that these cells at a strictly stage-specific fashion have a paracrine inhibitory effect on Sertoli cell inhibin alpha gene expression. Inhibin alpha mRNA level also correlates closely to the follicle-stimulating hormone-stimulated cAMP production during the cycle of the seminiferous epithelium, but does not seem to have a correlation to spermatogonial DNA synthesis.

Animals↗
PubMed1991

Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6).

Six different insulin-like growth factor binding proteins (IGFBPs) have been identified by molecular cloning of their cDNAs from rat and human tissues and designated as IGFBP-1, -2, -3, -4, -5 and -6. The total number of amino acid residues for the mature rat BPs ranges from 201 for IGFBP-6 to 270 for IGFBP-2, while the human homologs range from 216 for IGFBP-6 to 289 for IGFBP-2. Except for IGFBP-6, all rat and human IGFBPs contain 18 homologous cysteines; twelve are located at the N-terminal and span approximately one-third of the total amino acid sequence, while the remaining six are distributed at the C-terminal and span the last one-third of the protein sequence. Both rat and human IGFBP-4 possess two extra cysteines at the mid-region of the molecule. By contrast, rat and human IGFBP-6 contain only 14 and 16 cysteines, respectively. Absence of the two and four cysteines in the N-terminal region in the human and rat IGFBP-6 resulted in the deletion of the invariant Gly-Cys-Gly-Cys-Cys sequence which is present in all the other five IGFBPs. Both rat and human IGFBP-3 possess multiple N-linked glycosylation sites at the mid-region of the molecule, which accounts for their apparent molecular size being larger than the calculated molecular weight, based on the amino acid sequence. One potential N-linked glycosylation site is located at the mid-region of rat and human IGFBP-4, whereas only human but not rat IGFBP-6 possesses one N-linked glycosylation site at the extreme C-terminal of the molecule. An RGD sequence is found in the C-terminal of IGFBP-1 and -2. In this short review, updated information on the structural identification and molecular cloning of the six IGFBPs will be presented. In addition, the potential regulation of the BPs at the transcriptional and translational levels will be discussed.

Amino Acid Sequence↗
PubMed1991

Follistatin binds to both activin and inhibin through the common subunit.

Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of FSH release from pituitary cell culture. In addition to their effects on FSH synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin. Since activin binds to follistatin, it is imperative to determine the nature of the activin/follistatin binding complex. Moreover, because inhibin contains a beta-subunit derived from activin, it is important to determine whether inhibin will also bind follistatin. Using a double-ligand blotting technique, we have determined that activin-A has two binding sites for follistatin, whereas inhibin-A has only one binding site for follistatin. Therefore, these results suggest that follistatin binds to both activin and inhibin through the common beta-subunit.

Activins↗
PubMed1991

Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines.

A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology.

Amino Acid Sequence↗
PubMed1991

Cyclic changes in follistatin messenger ribonucleic acid and its protein in the rat ovary during the estrous cycle.

The purpose of this research was to characterize the localization of follistatin mRNA and protein in the adult rat ovary during the 4-day estrous cycle. Analysis of ovarian sections using in situ hybridization and immunohistochemistry demonstrated the presence of follistatin messenger RNA (mRNA) and its protein in granulosa and luteal cells; no follistatin (message or protein) was detected in any of the other ovarian cell types. An important observation was that the intensity of follistatin signals changed during granulosa differentiation and the estrous cycle. During folliculogenesis, the first detectable hybridization signal appeared in the granulosa cells of secondary follicles, but the signal was weak. However, when a preantral follicle reached the early tertiary stage (beginning antrum formation), the message signal was very strong, being expressed in all granulosa cells of all such follicles (300-400 microns in diameter). In atretic follicles, follistatin mRNA was localized to granulosa cells, but only during the early stages. The above hybridization pattern of follistatin mRNA in prenatral and atretic follicles appeared constant throughout the estrous cycle. Interestingly, immunohistochemistry studies showed that the follistatin protein was detected only in certain follicles, being restricted to those which were healthy. On the morning of estrus, the follistatin protein was localized to a subpopulation of early tertiary follicles, presumably the dominant follicles selected to ovulate in the next cycle. As the dominant preovulatory follicles matured through diestrus and proestrus, the follistatin mRNA and protein signals appeared more intense in the granulosa cells. After ovulation, the hybridization and immunohistochemical signals continued to be strong in the newly formed corpora lutea on estrus morning. After luteolysis on diestrus-I, neither the follistatin message nor the protein was detectable in the corpora lutea. In conclusion, these results suggest that the follistatin message is present in all the granulosa cells of every developing follicle throughout the estrous cycle, but the follistatin protein appears to be present in only the selected dominant follicles. Accordingly, the possibility that follistatin might be an important regulatory molecule for selection/atresia should be considered.

Animals↗
PubMed1991

Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin.

Follistatin is a glycosylated monomeric protein originally isolated from ovarian follicular fluid based on its ability to specifically inhibit pituitary FSH release. To further explore the physiological role of follistatin, we have expressed recombinant human follistatins with 315 (rhFS-315) and 288 (rhFS-288) amino acids in Chinese hamster ovary cells under the control of the simian virus-40 promoter. The two types of FS originated from alternatively spliced mRNAs and rhFS-315 differed from rhFS-288 by having an extra 27-amino acid sequence at the carboxyl-terminal. The yield of the purified rhFS-315 and rhFS-288 after a single step of affinity chromatography on an activin-coupled Affi-Gel column was 3-5 mg/liter conditioned medium. Using the rhFS-315 and rhFS-288 as molecular mass markers, Western blotting with FS carboxyl-terminal-specific antibodies demonstrated that the majority of native FS isolated from porcine ovarian follicular fluid was neither FS-315 nor FS-288, but was composed of 300 amino acids in various forms of glycosylation. This finding is consistent with our earlier results obtained from tryptic peptide fragment analysis of native FS. Only a very small percentage (less than 1%) of native porcine FS was FS-288. In cultures of rat anterior pituitary cells, rhFS-315 (ED50, 115.2 +/- 16.2 pM) is equipotent to native porcine FS (ED50, 86.7 +/- 14.1 pM) on the suppression of FSH release, but, surprisingly, rhFS-288 (ED50, 9.6 +/- 2.2 pM) is 8-10 times more potent than the native protein, similar to the potency of inhibin-A (ED50, 8.6 +/- 0.9 pM). Interestingly, when the in vivo FSH-suppressing activity of rhFS-288 was compared to that of inhibin-A in 1-week ovariectomized adult rats, it was found that rhFS-288 was more potent and longer acting than inhibin-A. Hence, these results indicate that FS-288 is probably one of the most potent natural FSH suppressors.

Amino Acid Sequence↗
PubMed1991

Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3, and 4) in the rat ovary.

We investigated the possible presence of the mRNAs encoding four insulin-like growth factor-binding proteins (IGFBP-1, -2, -3, and -4) in the rat ovary. It has been demonstrated previously by Northern analysis that adult rat ovaries contain mRNA transcripts for IGFBP-2 and -3. Here we show by Northern analysis that adult rat ovaries contain mRNA for IGFBP-4, but the mRNA for IGFBP-1 was below the limit of detection. Using in situ hybridization, IGFBP-1 mRNA was not detected in any of the ovaries tested. The IGFBP-2 mRNA was localized specifically in thecal interstitial cells (TIC) and secondary interstitial cells of all ovaries. The IGFBP-2 signal was very strong in TIC of all Graafian follicles (healthy and atretic) and very strong in all secondary interstitial cells located in different regions of the ovary, but weak in TIC of preantral follicles. The IGFBP-3 hybridization signal was localized specifically to some corpora lutea. Here the hybridization single for IGFBP-3 was strong and distributed throughout the corpus luteum, strong but localized to only some luteal cells, or not detectable. The IGFBP-4 mRNA was localized almost exclusively to granulosa cells of atretic follicles. The intensity of the IGFBP-4 signals appeared to increase as the level of atresia increased. In some cases, IGFBP-4 signals were detected in the TIC, but they were weak and variable. These results show that the mRNAs for IGFBP-2, -3, and -4 are localized to the interstitial cells, corpora lutea, and atretic granulosa cells, respectively. The tissue-specific synthesis of IGFBP subtypes in specialized ovarian cells provides an excellent system to study the manner in which IGFBP synthesis is controlled and their potential role as an autocrine and paracrine factors.

Animals↗
PubMed1991

Isolation and molecular cloning of insulin-like growth factor-binding protein-6.

Insulin-like growth factors (IGFs) together with their binding proteins (BPs) are potential regulators of folliculogenesis in mammalian ovary. To identify the various species of IGFBPs present in the ovary, we have undertaken a comprehensive purification scheme using gel filtration, ligand-affinity chromatography, and several steps of reverse phase HPLC to isolate all of the BPs in pig ovarian follicular fluid. Our effort yielded five distinct IGFBPs, and upon analysis, they were found to correspond to the previously identified human and rat IGFBP-2, -3, -4, -5, and -6. IGFBP-1 was not found in the pig ovarian follicular fluid under our experimental procedure. Of the six known classes of IGFBPs, the complete primary structures of the first five have been determined, but not IGFBP-6. Using amino acid sequence information from a tryptic fragment of pig IGFBP-6 to prepare a probe, cDNA clones encoding rat and human IGFBP-6 have been isolated and characterized. The deduced amino acid sequence revealed that rat IGFBP-6 contains 201 amino acids with a calculated mol wt of 21,461, while the human homolog contains 216 amino acids with a calculated mol wt of 22,847. In addition, a distinctive feature of human and rat IGFBP-6 is that they lack, respectively, two and four of the 18 homologous cysteines that are present in all other five IGFBPs. The missing cysteines in IGFBP-6 resulted in the absence of the invariant Gly-Cys-Gly-Cys-Cys sequence in the amino-terminal region of the molecule. Human IGFBP-6 possesses a single Asn-linked glycosylation site near the carboxyl-terminal, whereas no potential Asn-linked glycosylation sites are present in the rat sequence. A single 1.3-kilobase IGFBP-6 mRNA was detected by Northern analysis in all rat tissues examined, including testis, intestine, adrenal, kidney, stomach, spleen, heart, lung, brain, and liver, indicating that this BP is a ubiquitous protein. The chromosome location of the IGFBP-6 gene in human has been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that it is located on chromosome 12.

Amino Acid Sequence↗
PubMed1990

Structural characterization of a follicle-stimulating hormone action inhibitor in porcine ovarian follicular fluid. Its identification as the insulin-like growth factor-binding protein.

Recently, an inhibitory polypeptide that could block the follicle-stimulating hormone-induced estradiol and progesterone production in rat ovary granulosa cells has been isolated from porcine ovarian follicular fluid. Amino-terminal sequence analysis of the purified inhibitor suggests that it could be the porcine congener of the 53-kDa subunit of the growth hormone-dependent insulin-like growth factor binding protein (IGF-BP3). Using amino acid sequence information derived from the purified inhibitor to construct oligonucleotide probes, we have now identified the complementary deoxyribonucleic acids (cDNAs) encoding the inhibitory polypeptide from a porcine liver and a porcine ovary library. The nucleotide and predicted amino acid sequences revealed that the cDNAs indeed encode the porcine homolog of the recently characterized human IGF-BP3. The mature polypeptide consists of 266 amino acids, which is 2 amino acids longer than the human sequence. Between the two species, there are 42 amino acid substitutions, but the 18 cysteines and the three Asn-linked glycosylation sites are totally conserved. A single mRNA species of 2.6 kilobases encoding the IGF-BP3 was detected in porcine gonadal, brain, and liver tissues by Northern analysis.

Amino Acid Sequence↗
PubMed1990

Regulation of inhibin alpha- and beta B-subunit mRNA levels in rat Sertoli cells.

The hormonal regulation of the expression of the inhibin alpha-subunit and beta B-subunit genes was studied in cultured rat Sertoli cells. The alpha-subunit mRNA level increased during incubation of the cells in the presence of follicle-stimulating hormone (FSH), reaching maximal levels within 1.5 h. This stimulation was mimicked by addition of dibutyryl-cyclic AMP, indicating that FSH action on the alpha-subunit gene is exerted via cyclic AMP. Inhibition of translation by cycloheximide (CX) caused upregulation of the alpha-subunit mRNA, and did not block the effect of FSH on the level of this mRNA. In FSH-stimulated cells, the half-life of the alpha-subunit mRNA was 6 h, and this half-life was prolonged by inhibition of transcription using actinomycin D (AD). It is concluded that the effect of FSH on alpha-subunit mRNA expression represents a direct effect on the alpha-subunit gene, and that alpha-subunit mRNA levels are influenced by a short-lived mRNA destabilizing protein. The levels of two beta B-subunit mRNAs (4.2 kb and 3.5 kb) were not affected by FSH or dbcAMP. However, these mRNAs were also upregulated by CX treatment. Experiments using AD showed that the 4.2 kb mRNA is less stable than the 3.5 kb mRNA. The differential regulation of the inhibin alpha- and beta B-subunit mRNAs is discussed.

Animals↗
PubMed1990

Novel ovarian regulatory peptides: inhibin, activin, and follistatin.

Based on the extensive amount of research on inhibin and related polypeptides accomplished during the past 5 years, the inhibin concept put forth more than 50 years ago has not only become well established but also more complex than originally imagined. The closed-loop feedback mechanism of ovarian inhibin and pituitary FSH has been joined by possible "inhibin-like" actions of follistatin and FSH-stimulatory effects of activin. In addition, in vitro experiments suggest possible autocrine and paracrine functions for the gonadal polypeptide hormones. Figure 3 shows a simplistic diagram summarizing our current understanding of inhibin/activin and follistatin action along the hypothalamic-pituitary-gonadal axis. Hopefully, research in the coming years will allow us to remove the many question marks still remaining but will undoubtedly add others.

Activins↗
PubMed1990

Molecular cloning of the cDNAs encoding a novel insulin-like growth factor-binding protein from rat and human.

cDNA clones encoding a novel insulin-like growth factor-binding protein (IGFBP) purified from rat serum and human bone cell-conditioned medium have been isolated from rat liver and human placenta, liver, and ovary cDNA libraries. The deduced amino acid sequences of the cDNAs revealed a mature polypeptide consisting of 233 amino acids for the rat, while the human structure contains an additional four-amino acid sequence in the middle region of the molecule. This protein, now proposed to be named IGFBP-4, contains two extra cysteines compared with the previously characterized IGFBP-1, -2, and -3, but the alignment of the remaining 18 cysteines is conserved across the four IGFBPs. Amino acid sequence comparison among the four binding proteins within the rat species demonstrated that both the amino- and carboxy-terminal one thirds of the molecules are highly conserved, while the middle one third region, where no cysteines are present except for the two that exist in IGFBP-4, is the most divergent. The overall sequence homology among the four rat IGFBPs is very similar (53-59%), suggesting that their individual genes diverged from a single ancestral gene at about the same evolutionary time point. Northern analysis of the IGFBP-4 mRNA in rat tissue demonstrated that transcription of the IGFBP-4 gene is highly active in the liver, although a single 2.6-kilobase IGFBP-4 mRNA band was detectable in all tissues examined, including adrenal, testis, spleen, heart, lung, kidney, liver, stomach, hypothalamus, and brain cortex.

Amino Acid Sequence↗
PubMed1989

Complementary DNA structure of the high molecular weight rat insulin-like growth factor binding protein (IGF-BP3) and tissue distribution of its mRNA.

Insulin-like growth factors (IGFs) found in extracellular fluids are bound to specific binding proteins. Recently a high molecular weight IGF-binding protein (IGF-BP3) has been isolated from porcine ovarian follicular fluid based on its inhibition of follicle stimulating hormone-stimulated estradiol production in rat granulosa cells. The complete primary structure of the porcine IGF-BP3 was deduced by molecular cloning. Using the porcine cDNA as a probe, we have now isolated and characterized cDNAs encoding rat IGF-BP3 from a pregnant mare serum gonadotropin-stimulated ovarian library. The predicted amino acid sequence revealed a mature polypeptide consisting of 265 amino acids with 18 cysteines and 4 potential Asn-linked glycosylation sites. Northern analysis of the IGF-BP3 mRNA in rat tissues showed a single 2.6 kb band in liver, kidney, stomach, heart, adrenal, ovary, testis, spleen, lung, small and large intestine in varying amounts, but the message is below the limit of detection in hypothalamus and brain cortex.

Amino Acid Sequence↗