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Kazuhiko Imakawa

Publications and source records attributed to Kazuhiko Imakawa.

At least 19 recordsLinked to original sources
PubMed2024

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

UNLABELLED: Enzootic bovine leukosis (EBL), although eradicated in some European countries, is still the most common neoplastic disease of cattle, caused by the bovine leukemia virus (BLV). During the progression of EBL, BLV-infected cells clonally expand, and some of which result in tumor onset. The clonality of BLV-infected cells is generally evaluated with NGS or Sanger sequencing. Although these methods clearly distinguish EBL from non-EBL cases, the procedures are complex and not practical for routine veterinary diagnosis. In this study, we developed an amplified fragment length polymorphism (AFLP) analysis for BLV clonality assay (BLV-AFLP). This analysis uses restriction enzyme digestion to amplify the chimeric regions of BLV 3' linear transcribed region (LTR) and host genome through conventional polymerase chain reaction (PCR) and visualizes the results by gel-electrophoresis. The method was established using cattle samples representing different stages of the disease: BLV-uninfected, non-EBL, and EBL cattle. Non-EBL cattle showed smeared bands, indicating polyclonal proliferation, while EBL cattle showed distinct bands, indicating clonal expansion. The results of BLV-AFLP correlated well with those of previously reported methods, suggesting its efficacy in detecting clonal proliferation. The validation using blood samples of non-EBL cattle and tumor samples of EBL cattle confirmed that BLV-AFLP could effectively identify clonal proliferation in EBL samples. Moreover, the emergence of dominant clones in the tumor at later stages was successfully detected before EBL onset in some cattle, highlighting its sensitivity and potential for early detection. Overall, BLV-AFLP is suitable for practical use in the field, improving BLV management strategies and minimizing economic losses. IMPORTANCE: Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Leukemia Virus, Bovine↗
PubMed2006

Stability of casein mRNA is ensured by structural interactions between the 3'-untranslated region and poly(A) tail via the HuR and poly(A)-binding protein complex.

The maintenance of mRNA stability has emerged as a mechanism of post-transcriptional control. We demonstrate that beta-casein mRNA stability is dictated by the poly(A) tail and sequences in the 3'-UTR. An in vitro mRNA decay assay revealed that beta-casein mRNA with a long poly(A) tail had higher stability than that with a short poly(A) tail. The addition of poly(A) homopolymer and 3'-UTR cRNA as competitor induced rapid degradation of beta-casein mRNA. The interaction between full-length beta-casein mRNA and poly(A) homopolymer was inhibited by the addition of the 3'-UTR cRNA in the lysates, which indicates that one region of the 3'-UTR associates with the poly(A) tail through an RNA-protein interaction. The putative AU-rich element (ARE) is present at nt 897-905; deletion and mutation analysis showed that the ARE site was required for maintaining the stability of the beta-casein 3'-UTR. In the immunoprecipitation analysis, the poly(A)-binding protein (PABP) and the RNA-binding protein HuR were pulled down by 3'-UTR cRNA, and the absence of the ARE site reduced the binding of these proteins. These experiments further revealed that the rapid degradation of beta-casein mRNA was induced by incubation with HuR- and PABP-depleted RRLs. Collectively, our results suggest that beta-casein mRNA is protected from degradation by virtue of the structural interaction between the 3'-UTR and poly(A) tail via a protein complex of HuR and PABP.

3' Untranslated Regions↗
PubMed2006

Regulation of the ovine interferon-tau gene by a blastocyst-specific transcription factor, Cdx2.

Expression of ovine interferon-tau (oIFNtau), a factor essential for the process of maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast. However, the molecular mechanisms by which oIFNtau expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNtau gene transcription could be regulated through Cdx2 expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Human choriocarcinoma JEG3 cells were co-transfected with an oIFNtau (-654 base pair, bp)-luciferase reporter (-654-oIFNtau-Luc) construct and several transcription factor expression plasmids. Compared to -654-oIFNtau-Luc alone, transcription of the -654-oIFNtau-Luc increased more than 30 times when this construct was co-transfected with Cdx2, Ets-2, and c-jun. The degree of transcription decreased to 1/4 levels when the upstream region was reduced to -551 bp, and became minimal with further deletions; this was confirmed with the use of the reporter constructs with mutated c-jun, Ets-2, and/or Cdx2 sites. In trophoblast unrelated NIH3T3 cells, which do not support IFNtau gene transcription, the oIFNtau-Luc transcription was enhanced approximately eightfold when the cells were co-transfected with the Cdx2/Ets-2 or Cdx2/Ets-2/c-jun expression plasmids. These findings were confirmed by gel-shift assays examining Cdx binding site on the oIFNtau gene's upstream region, by immunohistochemical study identifying the presence of Cdx2 in day 15 and 17 ovine conceptuses, and by Western blot detecting Cdx2 in day 17 conceptuses. Our results indicate that oIFNtau gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNtau gene transcription by the trophectoderm.

Animals↗
PubMed2006

Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep.

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.

Animals↗
PubMed2006

Up-regulation of NOD1 and NOD2 through TLR4 and TNF-alpha in LPS-treated murine macrophages.

NOD1 (Card4) and NOD2 (Card15) are thought to be responsible for cytoplasmic defense against bacterial entry. To gain further knowledge about how their expressions are regulated in murine macrophages, we investigated the expression of NOD1 and NOD2 mRNAs after stimulation with various endotoxins, lipopolysaccharide, lipoteichoic acid and peptidoglycan. In macrophage RAW264.7 cells, the first and second rises in NOD1 and NOD2 mRNAs were observed at 2 hr and at 8-12 hr after endotoxin treatment. Increases in NOD1 and NOD2 mRNAs at 2 hr in lipopolysaccharide-treated RAW264.7 cells were reduced with the use of NF-kappaB inhibitor, caffeic acid phenethyl ester. In RAW264.7 cells, lipopolysaccharide-induced increases in NOD1 and NOD2 mRNAs were inhibited with anti-TLR4 antibody, and partially reduced in peritoneal macrophages obtained from TLR4-deficient mice. Furthermore, NOD1 and NOD2 mRNA expressions in RAW264.7 cells were increased by the treatment with proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), or IL-6. In TNF-alpha deficient macrophages, the expression of NOD molecules was minimal at 12 hr, and the second rise in NOD mRNA seen in lipopolysaccharide-treated RAW264.7 cells was inhibited with anti-TNF-alpha, but not with anti-IL-1beta or anti-IL-6 antibody. These observations suggest that immediate response of NODs to endotoxins could result from NF-kappaB activation via TLR signaling, whereas the second rise in NOD mRNAs might have resulted from TNF-alpha production possibly through NF-kappaB, TLR, and/or NOD signalings.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Progesterone initiates Wnt-beta-catenin signaling but estradiol is required for nuclear activation and synchronous proliferation of rat uterine stromal cells.

Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-beta. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-beta (0.2 microg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3beta (GSK-3beta) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3beta protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of beta-catenin in these stromal cells. Translocation of beta-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. Beta-catenin binding to TCF/LEF increased (P<0.05) in progesterone-pretreated uteri in response to estradiol. Progesterone stimulated the expression of the Wnt target gene urokinase plasminogen activator receptor (uPA-R) in the periluminal uterine stromal cells. The expression of uPA-R increased in progesterone-pretreated stromal cells in response to estradiol administration. Together, the results indicate that progesterone initiates Wnt signaling in the uterine stroma by down-regulating GSK-3beta. However, nuclear translocation of beta-catenin and sufficient complex formation with TCF/LEF to activate stromal cell cycle entry requires estradiol. Stimulation of a uterine stromal cell line to proliferate and differentiate resulted in beta-catenin accumulation, suggesting that endocrine-dependent Wnt signaling controls proliferation and differentiation (decidualization).

Animals↗
PubMed2005

Effects of progranulin on blastocyst hatching and subsequent adhesion and outgrowth in the mouse.

Using cDNA microarray methodology, we have shown previously that transcripts of progranulin gene (Grn, also known as acrogranin), a recently identified autocrine growth factor, were upregulated in mouse blastocysts adhered to the filter membrane in an in vitro-culture system. In the present study, we investigated the expression and effects of progranulin on blastocyst hatching, adhesion, and embryo outgrowth during the peri-implantation period in the mouse. During this period, substantial amounts of Grn mRNA were present in both inner cell mass (ICM) and trophectoderm. Progranulin was localized exclusively to the surface of the trophectoderm in early and pre- and postadhesion blastocysts as well as in trophoblast cells and ICM of outgrowth embryos, being secreted as a single, 88-kDa form into the surrounding medium. NIH3T3 cells that had been transfected with a progranulin expression construct secreted the 88-kDa form of the protein, from which a 68-kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant progranulin promoted blastocyst hatching, adhesion, and outgrowth, whereas rabbit anti-mouse progranulin immunoglobulin G reduced the incidence of blastocyst hatching, adhesion, and outgrowth. Studies of bromodeoxyuridine incorporation and immunodissection of the ICM revealed that progranulin was effective on the trophectoderm but not on the ICM. These results indicate that progranulin is an important factor for the processes of blastocyst hatching, adhesion, and outgrowth, and they suggest that the effects of progranulin on blastocyst adhesion and outgrowth may have been triggered by the previous action of progranulin to induce hatching of the blastocysts.

Animals↗
PubMed2005

Regulation of embryo outgrowth by a morphogenic factor, epimorphin, in the mouse.

Conceptus implantation to the uterine endometrium represents a complex series of events, including synchronized development of conceptus and uterus through up- and/or down-regulation of numerous gene products. In a previous study using the DNA microarray technique, we had discovered evidence that increase in a transcript for mesenchymal morphogen, epimorphin, was noted as the conceptus attached to the matrix in vitro (Qin et al., 2003). In the present study, the expression and potential function of epimorphin in developing conceptuses was investigated through the use of reverse transcription-polymerase chain reaction (RT-PCR), whole mount in situ hybridization/immunohistochemistry, and in vitro blastocyst culture. RT-PCR and in situ hybridization analysis revealed that epimorphin mRNA was expressed weakly in murine conceptuses during early developmental stages (1 cell to post-adhesion blastocyst stages) and higher levels of epimorphin transcripts were observed in both inner cell mass (ICM) and trophectoderm of outgrowing blastocysts. Immunohistochemical analysis confirmed that epimorphin was localized in outgrowing trophoblast cells and ICM. Treating blastocysts in culture with a 115 kDa form of recombinant epimorphin promoted trophoblast outgrowth (P < 0.05), but a 34 kDa form of recombinant epimorphin had no effect. Treatment with a function inhibitor, rat anti-mouse epimorphin IgM, reduced the number of embryos progressing to blastocyst outgrowth to the levels similar to those observed with plain culture medium. Reverse transcription-polymerase chain reaction (RT-PCR) analysis also revealed that epimorphin increased the expression of a trophoblast cell differentiation marker, placental lactogen-1 (PL-1), mRNA (P < 0.01). These results suggest that epimorphin is involved in trophoblast outgrowth, a process required for conceptus implantation into the endometrium.

Animals↗
PubMed2005

Different levels of ovine interferon-tau gene expressions are regulated through the short promoter region including Ets-2 binding site.

Regulation of interferon-tau (IFNtau) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNtau (oIFNtau) gene sequences characterized, approximately 75% of oIFNtau transcripts expressed in utero is derived from oIFNtau-o10 gene and amounts of transcripts from other oIFNtau genes such as oIFNtau-o8 or oIFNtau-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNtau-o10 and oIFNtau-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNtau genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 5'-upstream regions of these oIFNtau genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNtau-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNtau-o10 gene inserted into the oIFNtau-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNtau-o8/o2-reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNtau-o10 gene was required for oIFNtau-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNtau-o10 and oIFNtau-o8/o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNtau gene expressions seen in utero.

Animals↗
PubMed2005

Changes in immune cell distribution and IL-10 production are regulated through endometrial IP-10 expression in the goat uterus.

PROBLEM: Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon-gamma (IFN-gamma)-inducible protein 10 kDa (IP-10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP-10 during the period of pregnancy establishment. METHOD OF STUDY: Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of IP-10, IFN-gamma, tumor necrosis factor-alpha, interleukin-10 (IL-10), CXCR3 mRNA and leukocyte cell surface markers, CD4, CD8, CD11b and CD45 mRNA during the caprine early pregnancy was investigated. The ability of IP-10 to stimulate peripheral blood mononuclear cells (PBMCs) migration was demonstrated using a chemotaxis assay. Changes in migration of PBMCs' immune cell population and cytokine expressions with IP-10 stimulation were investigated using flow cytometry and RT-PCR respectively. RESULTS: Levels of IP-10, IL-10, CD4 and CD11b mRNA, and the number of CD4 and CD11b positive cells in pregnant goat endometrium were higher than those of cyclic goat endometrium. Migration of PBMCs was stimulated by recombinant caprine IP-10, and the effect was significantly reduced by neutralization with the use of an anti-IP-10 antibody. In the flow cytometric and RT-PCR analyses, migrated cells stimulated by IP-10 increased the expression of IL-10 and CD11b mRNA. Furthermore, IP-10 could stimulate the expression of IL-10 mRNA from PBMCs. CONCLUSION: Endometrial chemokine IP-10 could regulate IL-10 production by resident and possibly migrated cells expressing CD11b, probably natural killer cells, and these changes may result in immune environments of the uterus suitable for conceptus implantation in ruminants.

Animals↗
PubMed2005

Intestinal gene expression in TNBS treated mice using genechip and subtractive cDNA analysis: implications for Crohn's disease.

So far it has proven difficult to identify a causative gene(s) or gene product initiating the events that lead to inflammation of the intestinal mucosa and, ultimately, progression to Crohn's disease (CD), an inflammatory bowel disease. However, gene transcripts identified in the intestine of trinitrobenzene sulfonic acid (TNBS)-treated mice might suggest a clue, and even represent candidate genes leading to inflammation and mucosal damage, and to subsequent fibrosis. In the present study, DNA microarray (13000 transcripts) methodology was applied to mucosal RNA extracted from TNBS-treated mice, some transcripts of which were validated via cDNA subtraction and RT-PCR analyses. Intestinal biopsy samples from CD patients were then analyzed using cDNA mini-array (1300 cDNAs), focusing on gene transcripts associated with cancer and immunity. Mini-array results revealed transcript changes similar and also dissimilar to those found from the DNA microarray analysis. These changes, previously known or newly identified, possibly occurring during the initial and progressive stages of inflammatory conditions may provide a clue to identify marker transcripts and/or targets for the development of future gene therapy.

Animals↗
PubMed2004

Human keratin 14 driven HPV 16 E6/E7 transgenic mice exhibit hyperkeratinosis.

Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis.

Animals↗
PubMed2004

Increase in DNA methylation downregulates conceptus interferon-tau gene expression.

Expression of ovine interferon-tau (oIFNtau) genes, essential for the maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast and is not detected in any other cell types or tissues. Substantial secretion of oIFNtau starts on day 12-13 of pregnancy (day 0 = day of estrus), reaches the highest on day 16-17, and then declines rapidly. Ovine IFNtau mRNA, on the other hand, reaches the highest level on day 14 of pregnancy, 2-3 days before peak production of the protein. In this study, day 14 and 17 conceptuses treated with 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, were cultured in vitro and only day 17, not day 14, conceptuses resulted in upregulation of oIFNtau gene expression. To gain insight into the molecular mechanism of oIFNtau gene downregulation, the methylation status within 1 kb of the 5'-flanking region of oIFNtau-o10 gene was investigated: CpG dinucleotides of this gene in day 14 ovine conceptuses were hypomethylated compared to day 20 conceptuses or other tissues. In vitro methylation of oIFNtau-o10-reporter constructs caused suppression of reporter activity in transient transfections. Cotransfection of methyl-CpG-binding protein (MeCP2) with the reporter construct elicited further suppression of the reporter activity. In electrophoretic mobility shift assay (EMSA), patterns of shifted bands did not show much difference between methylated and unmethylated probes in distal regions, but exhibited differences in the proximal region of upstream sequences of the oIFNtau gene. These results provide evidence that changes in the degree of DNA methylation could be one of the major mechanisms leading to downregulation of the oIFNtau-o10 gene during early gestation, and possibly its silencing in nonconceptus tissues.

Animals↗
PubMed2004

Pre-implantation conceptus and maternal uterine communications: molecular events leading to successful implantation.

Implantation, a critical step for mammals in establishing pregnancy, requires successful completion of sequential events such as maternal uterine development, conceptus development and attachment, and placental formation. To reach the stage of placental formation, synchronized development of the conceptus and uterus throughout the implantation period is absolutely required. A number of factors expressed at the uterine endometrium and/or conceptus, which are associated with peri-implantation development, have been identified. In addition to a temporal and spatial expression of these factors, their roles in intra- and inter-cellular interactions make it difficult to fully understand physiological roles played during the critical period. This paper focuses on early conceptus development, maternal preparation for implantation and uterine-conceptus communication during the pre-implantation period, rather than the subsequent events such as conceptus attachment to the maternal endometrium. New aspects of pre-implantation processes are evaluated through simultaneous expressions of transcription factors as they possibly regulate the complex processes of implantation events in murine species and ruminant ungulates.

Animals↗
PubMed2004

A potential live vector, foamy virus, directed intra-cellular expression of ovine interferon-tau exhibited the resistance to HIV infection.

Interferon-tau (IFN-tau), produced by the embryonic trophectoderm, is a member of type I IFNs required for the establishment of pregnancy in the ruminant ungulates. Although this IFN possesses antiviral activity similar to other type I IFNs, the effectiveness of IFN-tau as an antiviral agent has not been well characterized. To investigate possible antiviral effects of ovine IFN-tau (oIFN-tau), oIFN-tau-GST fusion protein was expressed in E. coli BL21, from which the purified protein isolated possessed anti-viral activity. An apathogenic human foamy virus (hFV) was then used to establish a potential recombinant live vector consisting of oIFN-tau cDNA sense (+) or antisense (-) sequence, oIFN-tau(+)/hFV or oIFN-tau(-)/hFV, respectively. Human hematopoietic and other mammalian cell lines that had been transduced with hFV vector consisting of no oIFN-tau, oIFN-tau(+)/hFV or oIFN-tau(-)/hFV construct were cultured initially for 12 days, and three of cell lines were then maintained for up to 90 days. These cells with oIFN-tau expression directed by hFV exhibited the in vitro cytopathic effect minimally. Transduced cell lines that had been cultured for 90 days were subjected to studies on human immunodeficiency virus type-1 (HIV-1) infection, which was measured with infectivity of viral particles resulted from the GFP inserted T-cell tropic HIV SF2 or macrophage tropic HIV SF162: the number of HIV-1 positive cells was reduced by the hFV driven-intra-cellular oIFN-tau expression. Since oIFN-tau/hFV transduced cells exhibited the resistance to HIV-1 infection and/or replication, oIFN-tau could be considered as one of effective antiviral agents against HIV-1. These results suggest that the hFV genome could be an effective recombinant live vector for the expression of a targeted gene in various cell types.

Animals↗
PubMed2003

Regulation of blastocyst migration, apposition, and initial adhesion by a chemokine, interferon gamma-inducible protein 10 kDa (IP-10), during early gestation.

For a pregnancy to be established, initial apposition and adhesion of the blastocyst to maternal endometrium must occur in a coordinated manner; however, a key factor(s) that mediates the trophoblast cell migration and attachment to the apical surface of the endometrium has not been identified. In this study, we examined the effect of an endometrial chemokine, interferon-gamma-inducible protein 10 kDa (IP-10), on conceptus migration to the endometrial epithelium. We first studied endometrial IP-10 mRNA expression, which was localized in the subepithelial stromal region, and detected the protein in the uterine flushing media during early pregnancy. Expression of IP-10 mRNA by the endometrium of cyclic animals was stimulated by the addition of a conceptus factor interferon-tau (IFN-tau). Immunofluorescent analysis revealed that IP-10 receptor, CXCR3, was localized in the trophoblast cells, to which biotinylated-recombinant caprine IP-10 (rcIP-10) bound. Chemotaxis assay indicated that rcIP-10 stimulated the migration of trophoblast cells, and the effects of rcIP-10 were neutralized by the pretreatment with an anti-IP-10 antibody. Adhesive activity of trophoblast cells to fibronectin was promoted by rcIP-10, and the effect was inhibited by the use of anti-IP-10 antibody. Further adhesion experiments demonstrated that binding of trophoblast cells to fibronectin was completely inhibited by a peptide of the Arg-Gly-Asp (RGD) sequence, which binds to integrins alpha5beta1, alphaVbeta1, alphaVbeta3, and alphaVbeta5, whereas non-binding peptide containing Arg-Gly-Glu (RGE) had minimal effects. More importantly, rcIP-10 promoted the adhesion of trophoblast cells to primary cells isolated from endometrial epithelium. Furthermore, rcIP-10 stimulated the expression of integrin alpha5, alphaV, and beta3 subunit mRNA in trophoblast cells. These findings suggest that endometrial IP-10 regulates the establishment of apical interactions between trophoblast and epithelial cells during early gestation.

Animals↗
PubMed2003

Coactivator CBP in the regulation of conceptus IFNtau gene transcription.

Studies of ovine interferon-tau (oIFNtau) gene regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, have been carried out, but a definitive mechanism for its spatial-temporal transcription has not been elucidated. Recently, specific binding regions for transcription factors AP-1 and Ets-2 on the oIFNtau gene were identified; however, a molecular mechanism by which these factors regulate oIFNtau gene transcription has not been characterized. In the present study, we investigated the potential relationship between AP-1 and Ets-2, and their association with a coactivator, cAMP-response element binding protein-binding protein (CBP), on oIFNtau gene transcription in a transient transfection system using human choriocarcinoma JEG3 cells. The oIFNtau gene promoter/enhancer (-654 to + 1 bases, wild type)-luciferase reporter construct (pGL3-654) or its mutant at the AP-1 or Ets-2 site was cotransfected with CBP (pRc/RSV-CBP) construct along with c-jun, c-fos, and/or Ets-2 expression plasmid. CBP enhanced transcription of the wild type oIFNtau-reporter construct; however, this coactivator had no effect on the oIFNtau-reporter construct with mutated AP-1 or Ets-2 site. Cotransfection of CBP with c-jun and/or Ets-2, but not with c-fos, further increased oIFNtau gene transactivation although amounts of c-jun and c-fos expression, resulting from expression vectors, were similar. In addition, CBP inhibitor adenovirus 12S E1A (E1A), but not the mutant of E1A without CBP binding domain (Delta2-36), suppressed oIFNtau gene transcription. These observations suggest that c-jun and Ets-2 are the most probable binding partners for CBP in the potentiation of oIFNtau gene transcription. Mol. Reprod. Dev. 65: 23-29, 2003.

Choriocarcinoma↗