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K Imakawa

Publications and source records attributed to K Imakawa.

At least 37 records · Page 2Linked to original sources
PubMed1993

A novel role for GM-CSF: enhancement of pregnancy specific interferon production, ovine trophoblast protein-1.

Transient and massive production of ovine trophoblast protein-1 (oTP-1) by preimplantation conceptuses seems to be a critical event required for the establishment of successful pregnancy. We have previously demonstrated that one of several oTP-1 genes is predominantly expressed between days 13 and 20 of pregnancy and that this oTP-1 gene contains an AP-1 site, a transcription enhancer element, in the 5'-flanking region. We have obtained evidence, indicating a linkage between cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and conceptus production of the trophoblast interferon (IFN), oTP-1. These are: 1) oTP-1 production (both polypeptide and mRNA) is enhanced by the addition of GM-CSF in vitro and 2) GM-CSF mRNA is localized in the luminal and glandular epithelium of the uterine endometrium. Based on these observations, we propose that the massive amounts of oTP-1 produced during the period of pregnancy establishment is stimulated at least in part by maternal GM-CSF.

Animals↗
PubMed1992

Expression of tumor necrosis factor-alpha in the rat ovary.

Tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) in rat ovaries was detected by Northern blot analysis and by reverse transcription-polymerase chain reaction (RT-PCR). Northern blot analysis of total cellular RNA (tcRNA) isolated from adult rat ovaries showed a single distinct band at approximately 2 kilobases, while rat white blood cells, used as control, also showed a second lower band at approximately 0.5 kilobases. TNF-alpha expression in ovaries was also detected by RT-PCR, using RNA-specific primers. Southern blot analysis of the RT-PCR products showed a single band of the expected 500 base pairs, from the ovary and white blood cells, using either tcRNA or polyA+ RNA. In order to validate the RT-PCR product, it was digested with restriction enzymes, HhaI, HindIII, and XhoI. The results indicate that the ovarian TNF-alpha mRNA does not contain major alterations with respect to the known structure of rat TNF-alpha mRNA. Ovarian RNA was also subjected to PCR without previous RT in order to amplify, if any, contaminating genomic DNA. A single band of approximately 900 base pairs was observed, which contained introns 1 and 2 (determined by restriction enzyme digestion). In summary, these results indicate that rat ovaries contain TNF-alpha mRNA which makes it a likely source of local TNF-alpha secretion. The possibility exists that ovarian cells and/or macrophages are the source of the ovarian TNF-alpha mRNA.

Animals↗
PubMed1992

Evidence for lipopolysaccharide binding in human granulosa-luteal cells.

We investigated whether human granulosa-luteal (GL) cells exhibited lipopolysaccharide (LPS)-binding protein, and the response of follicular aspirate cells to LPS in vitro. Follicular aspirates taken from a human in-vitro fertilization and gamete intra-fallopian-tube transfer programme were subjected to Percoll gradients in order to isolate an enriched population of GL cells. GL cells exhibited specific LPS-binding protein, detected by autoradiography of the cellular lysate on SDS-PAGE after the cells were specifically labelled with a radioiodinated, photoactivable and reducible LPS derivative. LPS binding to the cells was also detected by the appearance of immunofluorescence associated with the cellular membrane when incubated with a fluorescent conjugated LPS receptor antibody. Ninety-four per cent of the cells exhibiting immunofluorescent LPS-binding protein were also positive for the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase, as detected by cytochemistry. In order to detect a response to LPS, the enriched population of GL cells were cultured in vitro in the presence or absence of LPS; after 16 h of culture, tumour necrosis factor-alpha (TNF) mRNA was detected by reverse transcription-polymerase chain reaction and Southern blot analysis of the amplified cDNA. The expression of TNF mRNA was enhanced when the cells were cultured in the presence of LPS, which also significantly enhanced TNF secretion into the media during the 16-h period. These results reveal that GL cells exhibit LPS-binding protein and thus increased TNF secretion occurs in response to LPS in follicular aspirate cells. The source of ovarian TNF may be leukocytes, macrophages and/or GL cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Cloning and analysis of a gene encoding ovine interferon alpha-II.

A gene encoding ovine interferon alpha (IFN alpha) was identified from an ovine liver genomic library. Based on nucleotide and deduced amino acid sequencing analyses, this gene appears to code for an IFN of the alpha II family different from the ovine embryonic IFNs found to date.

Amino Acid Sequence↗
PubMed1991

Circulating concentrations of 17-estradiol influence pattern of LH in circulation of cows.

The objective of the research was to determine the relationship between circulating 17 beta-estradiol (E2) and secretion of luteinizing hormone (LH) in cows. A second objective was to determine if response to E2 was influenced by interval between ovariectomy and the start of E2 treatment. Thirty-one nulliparous cows 3 yr of age were randomly assigned to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age (long term), and the other 15 cows were ovariectomized at 36 mo of age (short term). At the time of ovariectomy of cows in the short term group, 11 cows in the short term group and 12 cows in the long term group were implanted subcutaneously with 1, 2 or 4 polydimethylsiloxane capsules containing E2. The other eight cows served as non-implanted controls (n = 4-short term, n = 4-long term). All cows were fitted with jugular vein catheters on day 29 of treatment, and on day 30 blood samples were collected at 12-min intervals for 6 hr. At the end of 6 hr, luteinizing hormone-releasing hormone (LHRH) was administered and blood sampling continued at 12-min intervals for an additional hour. Serum was analyzed for LH and E2. Variables of LH secretion analyzed were mean concentration, frequency of pulses, amplitude of pulses and maximum concentration after LHRH. There were no significant interactions for any of the variables of LH among cows ovariectomized for the long and short term. There was a significant linear increase in mean concentration of LH with increased circulating concentration of E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Slowed transcription and rapid messenger RNA turnover contribute to a decline in synthesis of ovine trophoblast protein-1 during in vitro culture.

Ovine trophoblast protein-1 (oTP-1) is produced in massive amounts by conceptuses during the Day-12-20-period of early pregnancy. The rate of production of oTP-1 declines during culture of conceptuses, however, suggesting a role for critical intrauterine factors for continued production. The present study was conducted to define the mechanism responsible for the in vitro decline in oTP-1 synthesis. Over 24 h culture, synthesis of oTP-1 was initially rapid but then declined such that there was little increase in either the protein or its antiviral activity after 12 h. By contrast, the release of total protein into the medium continued at an approximately linear rate for the entire 24-h period of culture. By using in situ hybridization to tissue sections and dot-blot analysis of tissue extracts with labeled cDNA probes, it was shown that the quantity of oTP-1 mRNA fell 3- to 5-fold during culture, whereas the amount of actin mRNA remained relatively constant. To determine whether this selective fall in levels of oTP-1 mRNA resulted from decreased transcription rate and/or high turnover rate of existing mRNA, conceptuses were either provided continuously with [3H]uridine for 24 h or labeled for 9 h and then exposed to medium enriched in unlabeled uridine and cytidine for a further 15 h. Specific incorporation of 3H into oTP-1 mRNA was assessed by hybridization to excess oTP-1 cDNA immobilized on nitrocellulose membranes. In the continuous labeling study, 3H in total RNA increased at an approximately linear rate for at least 18 h, whereas the content of 3H in oTP-1 mRNA peaked at 9 h and declined about 10-fold by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The polypeptides and genes for ovine and bovine trophoblast protein-1.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) have been strongly implicated as antiluteolytic agents and responsible for maternal recognition of pregnancy in sheep and cattle, respectively. Both are interferons (IFN) belonging to the IFN-alpha family, but their length (172 residues versus 166 for most IFN-alpha) places them in an unusual subclass (the IFN-alpha II). The various isoforms of oTP-1 and bTP-1 produced by trophoblast tissue appear to arise in part from translation of multiple mRNAs which are themselves the products of distinct genes. These genes, like those for other IFN-alpha, are without introns. However, the genes for oTP-1 and bTP-1 form a distinct subgroup within the IFN-alpha II on the basis of their overall primary sequences and the high conservation of the 3'-untranslated ends of their transcription units. The bTP-1 genes also differ from the bovine IFN-alpha II in the organization of the promoter regions upstream from the transcription start site. Nevertheless, computer-aided analysis of the primary polypeptide sequences of oTP-1 and bTP-1 indicates that the molecules are likely to have approximately the same shapes and dimensions as all other IFN-alpha molecules. It remains to be determined whether they have unique biological properties which distinguish them from other IFN-alpha molecules.

Amino Acid Sequence↗
PubMed1990

Expression of trophoblastic interferon genes in sheep and cattle.

The trophoblastic interferons ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1, respectively) have been implicated as mediators of maternal recognition of pregnancy in sheep and cattle. The objective of this study was to describe the onset and duration of gene expression for oTP-1 and bTP-1 in preimplantation ovine and bovine conceptuses by in situ hybridization and Northern analysis. Sections from paraffin-embedded ovine conceptuses, collected on Days 10, 11, 12, 13, and 15 of gestation (n = 1, 3, 3, 2, 2), and bovine conceptuses, collected on Days 12/13, 15/16, and 19 (n = 2, 4, 5), were hybridized to specific [35S]-labeled cDNA probes. Two different probes, one encompassing bases 442-918 and representing both coding and 3'-untranslated regions, and a second 3'-specific probe (bases 650-912) were used to detect oTP-1 mRNA. At all stages examined, oTP-1 mRNA was confined to trophectoderm of ovine conceptuses. Consistent with earlier studies, expression increased markedly at Day 13. oTP-1 mRNA was detected at low levels in seven of seven ovine conceptuses prior to Day 13 when the longer probe was employed. With the 3'-specific probe, however, oTP-1 mRNA was detected in only one of the seven ovine conceptuses prior to Day 13. Thus, although low amounts of oTP-1 mRNA may be present in ovine conceptuses prior to Day 13, massive induction of this mRNA occurs on Day 13 coincident with the initiation of maternal recognition of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon).

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Molecular cloning of the uteroferrin-associated protein, a major progesterone-induced serpin secreted by the porcine uterus, and the expression of its mRNA during pregnancy.

The uteroferrin(Uf)-associated basic proteins (UfAP) are a group of three (Mr = 42K, 48K, and 50K) antigenically related, basic glycoproteins secreted by the porcine uterus under the influence of progesterone (P4) which exist as heterodimers (Mr = 80,000) with the iron-binding acid phosphatase, Uf. Several UfAP cDNA clones from a day-60 pregnant pig uterine endometrial cDNA library have been cloned and sequenced. The UfAP mRNA is approximately 1400 bases long and has a single open reading frame of 1251 bases with two start codons at positions 64 and 79 from the 5'-end. UfAP mRNA content of the endometrium increases as pregnancy proceeds, reaching maximum levels around day 70 and then remaining relatively constant in late gestation (days 70 to 110). The pro-form of the UfAP minus signal sequence appears to be 392 amino acids in length and has four potential N-linked glycosylation sites Asn107, Asn197, Asn243, and Asn315. Comparison of the NH2-terminal sequences of the individual UfAP poly-peptides with the amino acid sequence deduced from the cDNA has indicated a series of at least four posttranslational proteolytic processing steps which generate the various molecular forms of the UfAP. The deduced amino acid sequence of UfAP shares considerable identity with several protease inhibitors and hormone-binding proteins that are members of the serpin superfamily of proteins. The UfAP amino acid sequence also exhibits about 55% sequence identity with the P4-induced uterine milk proteins (UTMP) of the sheep. Since the UfAP and UTMP share many biosynthetic and structural features that include site of biosynthesis in the endometrium, P4-responsiveness, the presence of the mannose 6-phosphate lysosomal recognition marker, and considerable sequence similarity, the UfAP and the UTMP may have homologous function which for both still remains obscure.

Acid Phosphatase↗
PubMed1990

Interferons at the placental interface.

The antiluteolytic factors secreted by sheep and cattle conceptuses are closely related structurally to alpha-interferons (IFN-alpha s). They are known as ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1), respectively. The mRNAs for oTP-1 and bTP-1 are transcribed from multiple genes and are the major translatable messages of Day 13-17 sheep conceptuses and Day 15-20 cattle conceptuses. The proteins belong to the 172-amino acid IFN-alpha II (or IFN-omega) subfamily and have the typical antiviral and antiproliferative properties of the 166-residue IFN-alpha Is. These embryonic interferons also bind to the IFN-alpha receptor, which is present in uterine endometrium in high concentrations, and can influence the production of prostaglandin F-2 alpha and the pattern of protein secretion in that tissue. Through use of in-situ hybridization procedures on tissue sections and Northern and dot blot analyses of extracted conceptus RNA, ovine oTP-1 mRNA has been shown to increase markedly around Day 13 and to decrease after about Day 15 of pregnancy. The mRNA is confined entirely to cells of the trophectoderm. Significant induction of mRNA that hybridizes to an oTP-1 cDNA occurs in response to exposure to polyI:polyC in Day 11 sheep blastocysts which normally have low levels of oTP-1 expression. However, the basis for induction in the normal progression of embryonic development remains unclear. The fact that preimplantation conceptuses of other species, e.g. pig, release substances with antiviral activity suggests that IFNs may have an important role in pregnancy that extends beyond the domestic ruminants.

Amino Acid Sequence↗
PubMed1989

Interferon production by the preimplantation sheep embryo.

Ovine trophoblast protein-1 (oTP-1), the major product secreted by the trophectoderm of the sheep conceptus between days 13 and 21 of pregnancy, is considered to mediate maternal recognition of pregnancy by maintaining the function of the corpus luteum. Its amino acid sequence has 40-55% identity with various mammalian interferons-alpha (IFN-alpha), and it has been shown to have antiviral activity. The present results confirm that oTP-1, which at days 15-17 of pregnancy is produced by a single embryo at more than 100 micrograms (greater than 1 million antiviral units) per day, is a functional IFN. A preparation of purified oTP-1 was made. Its amino-terminal sequence suggested that it consisted of a single homogeneous protein, so that its antiviral activity probably was not due to a contaminant. In a cytopathic effect inhibition assay with GBK-2 bovine cells challenged with vesicular stomatitis, its specific activity was 1.3 X 10(7) end point units/mg protein. It also protected GBK-2 cells against four other viruses, and A549 human cells against encephalomyocarditis virus. The antiviral activity was neutralized by an antiserum to human leukocyte IFN. Like human IFN-alpha, oTP-1 at concentrations as low as 10(-9) M inhibited the growth of GBK cells in culture and suppressed mitogen-stimulated incorporation of [3H]thymidine into ovine lymphocytes. Possible roles for oTP-1, functioning as an IFN-alpha during early pregnancy, are discussed.

Age Factors↗
PubMed1989

Complex binding of the embryonic interferon, ovine trophoblast protein-1, to endometrial receptors.

Ovine embryos produce an interferon (IFN)-alpha II in significant quantities during early pregnancy. This IFN, previously termed ovine trophoblast protein-1 (oTP-1), is a 172-amino-acid polypeptide which has been suggested to be the causal agent in maternal recognition of pregnancy in the ewe. Here we report the binding of oTP-1 and a recombinant bovine IFN-alpha I1 (rBoIFN-alpha I1; 165-166 amino acids long) to membrane preparations from ovine uterine endometrium. Both oTP-1 and rBoIFN-alpha I1 competed with each other for receptor binding. Based on Scatchard analysis, [125I]oTP-1 binding was determined to be complex and resolvable into a high-affinity (Kd = 3.8 x 10(-11) M, 30 fmoles/mg protein) and a low affinity (Kd = 1.7 x 10(-10) M; 96 fmoles/mg protein) component. Conversely [125I]rBoIFN-alpha I1 bound to only a single high-affinity receptor (Kd = 6.1 x 10(-11) M; 174 fmoles/mg protein). Cross-linking experiments using disuccinimidyl suberate revealed that [125I]oTP-1 associated with membrane polypeptides of two molecular weight classes (Mr 100,000 and 70,000), and could be displaced from both with rBoIFN-alpha I1. In contrast, [125I]rBoIFN-alpha I1 cross-linked to only the 100,000 Mr membrane polypeptide. These data provide evidence that the binding parameters of oTP-1 and rBoIFN-alpha I1 to endometrial receptors are different.

Animals↗
PubMed1989

Suppression of T-lymphocyte blastogenesis by ovine trophoblast protein-1 and human interferon-alpha may be independent of interleukin-2 production.

Cells derived from the trophoblast tissue of a day 15 sheep conceptus released substances that inhibit incorporation of [3H]thymidine into phytohemagglutinin (PHA)-stimulated ovine lymphocytes. This effect was partially reversed by addition of antiserum to ovine trophoblast protein-1 (oTP-1), a major secretory product of day 13-21 sheep conceptuses and a protein structurally and functionally related to alpha-interferons (IFN-alpha). Human IFN-alpha, unlike dexamethasone, inhibits phytohemagglutinin-induced lymphocyte blastogenesis without reducing interleukin-2 (IL-2) production by the cultures, and conditioned medium containing IL-2 does not promote [3H]thymidine incorporation into ovine lymphocytes when oTP-1 is present. Thus, oTP-1, by virtue of being an IFN, may have a local immunomodulatory role by selectively inhibiting the proliferative responses of certain maternal immune cells to IL-2.

Animals↗
PubMed1989

Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-1: a comparison with ovine trophoblast protein-1 and bovine interferon-alpha II.

Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18-19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5' end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% identical to that for a bovine interferon-alpha II (IFN alpha II). The highest conservation of sequence (greater than 90%) was noted in the 3'-untranslated sequences of the bTP-1 and oTP-1 cDNAs. The deduced amino acid sequence of bTP-1 shared 80% identity with oTP-1, between 45-55% with human, rodent, porcine, and bovine IFNs of the alpha 1 subfamily and about 70% with a bovine IFN alpha II. A single potential site for N-glycosylation was noted at Asn78. These results show that bTP-1, like its ovine counterpart oTP-1, is structurally related to the IFN alpha S. We suggest that these embryonic IFNs play a role in controlling immunoreactions at the trophoblast-uterus interface as well as triggering other maternal responses to pregnancy.

Amino Acid Sequence↗
PubMed1989

In situ localization of mRNA for the interferon, ovine trophoblast protein-1, during early embryonic development of the sheep.

The embryonic interferon, ovine trophoblast protein-1 (oTP-1), is considered to be the major protein signal by which the developing ovine conceptus communicates its presence to the mother in order to provide extension of luteal progesterone secretion critical for the establishment of pregnancy. The objective of the present study was to examine the distribution of mRNA for oTP-1 in developing ovine embryos by using in situ hybridization. A total of 11 ovine embryos were collected on days 11, 13, 15, 17, and 23 of gestation (n = 1, 2, 3, 3, and 2, respectively) and were subjected to either immediate paraformaldehyde fixation or culture for 24 h followed by fixation. Fixed embryos were embedded in paraffin and oTP-1 mRNA levels determined by in situ hybridization with a 35S-labeled cDNA probe specific for the 3'-untranslated region of the oTP-1 mRNA. Controls included parallel hybridizations with a 35S-labeled gamma-actin cDNA to detect actin mRNA (positive control) and with 35S-labeled plasmid cDNA (negative control). Hybridization signals were detected by autoradiography and quantified by computer-assisted video image analysis. Ovine TP-1 mRNA levels in tissue were low but detectable on day 11, rose 6.5-fold to peak concentrations on day 13, and declined in a linear fashion through day 23. A low, detectable signal was present in portions of chorionic tissue on day 23. Messenger RNA was localized solely to the trophectoderm and did not appear in the extraembryonic endoderm, yolk sac, and embryonic disc. The relative hybridization signal for actin mRNA was approximately 12-fold lower than that for oTP-1 mRNA on day 13. However, by day 17 oTP-1 and actin mRNA hybridization signals were similar. In conclusion, oTP-1 mRNA is localized in the trophectoderm of the developing embryo, being produced between days 11 and 23 of gestation with peak amounts produced per cell at approximately day 13 of gestation.

Animals↗