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B Kalionis

Publications and source records attributed to B Kalionis.

At least 19 recordsLinked to original sources
PubMed2006

Homeobox gene DLX4 expression is increased in idiopathic human fetal growth restriction.

Idiopathic fetal growth restriction (FGR) is often associated with placental insufficiency. Previously, we isolated and characterized homeobox gene DLX4 from the placenta and provided evidence that DLX4 may regulate placental development. Here, we have investigated whether DLX4 expression levels were altered in idiopathic FGR. FGR-affected placentae were collected based on strict clinical criteria. DLX4 mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Initial RT-PCR results showed a qualitative increase in DLX4 mRNA in both FGR-affected placentae and gestation-matched controls. Real-time PCR showed a 3-fold increase in DLX4 mRNA levels in FGR-affected placentae compared with gestation-matched controls (P < 0.005). Western immunoblotting using a rabbit DLX4 polyclonal antibody revealed significantly increased levels of DLX4 protein in term FGR-affected placentae compared with term controls [5500.1 +/- 21.8 (n = 10) versus 3533.2 +/- 22.4 (n = 10); P < 0.001]. Qualitative immunohistochemical analyses of term placentae showed moderately increased immunoreactivity for DLX4 antigen in the FGR-affected placentae in syncytiotrophoblasts, residual cytotrophoblast cells and endothelial cells of the fetal capillaries compared with gestation-matched control term placentae. We conclude that the increased expression of homeobox gene DLX4 may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae.

Adult↗
PubMed2006

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

Chloride Channels↗
PubMed2006

Homeobox genes are differentially expressed in macrovascular human umbilical vein endothelial cells and microvascular placental endothelial cells.

Angiogenesis is fundamental to normal placental development. Aberrant angiogenesis within the placental terminal villi is a characteristic of significant placental pathologies and includes structural and vascular abnormalities as well as altered endothelial cell function, which substantially impacts on maternal-fetal exchange. Homeobox gene transcription factors regulate vascular development in embryonic and adult tissues, but their role in the placental microvasculature is not well known. In this study, we isolated and enriched human placental microvascular endothelial cells (PLEC) by a perfusion-based method and compared homeobox gene expression between PLEC and macrovascular human umbilical vein endothelial cells (HUVEC). Reverse transcriptase PCR detected mRNA expression of homeobox genes DLX3, DLX4, MSX2, GAX and HLX1 in both PLEC and HUVEC. DLX4 and HLX1 have not been previously detected in PLEC and with the exception of GAX, none of these homeobox genes have been previously identified in HUVEC. There was lower expression of HLX1 mRNA in HUVEC compared with PLEC. Using real-time PCR analysis PLEC HLX1 mRNA expression relative to housekeeping gene GAPDH was 0.9+/-0.06 fold of the calibrator (n=6) versus 0.2+/-0.06 (n=6) for HUVEC, p<0.001. These data provided evidence of heterogeneity in homeobox gene expression between microvascular PLEC and macrovascular HUVEC that most likely reflects significant differences in endothelial cell function in the two different cellular environments.

Endothelial Cells↗
PubMed2006

Trophoblast differentiation: progenitor cells, fusion and migration -- a workshop report.

Challenge lies ahead in unravelling the role played by trophoblast and its repertoire of expressed genes in normal human placental development, growth and pathology. Specific technical advances will clearly be required for characterisation of function. In particular, improvements in our repertoire of in vitro models are needed before many of the key questions can be answered. Recent advances in the study of human trophoblast differentiation are discussed.

Cell Communication↗
PubMed2005

Expression of GLUT12 in the fetal membranes of the human placenta.

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.

Adult↗
PubMed2005

Fetal growth restriction is associated with increased apoptosis in the chorionic trophoblast cells of human fetal membranes.

The aim of the study was to determine the incidence and spatial distribution of apoptotic cell in fetal membranes obtained from human pregnancies complicated with fetal growth restriction (FGR) for which there was no established cause. Fetal membrane samples from normal (n=10) and FGR-affected (n=10) pregnancies were collected and stored following delivery. The incidence of apoptosis and the number of apoptotic cells in normal and FGR-affected fetal membranes were determined using immunohistochemistry and a monoclonal antibody for neo-epitope of cytokeratin-18, M30. The level of apoptotic proteins in FGR-fetal membranes compared to the normal tissue was determined using Western immunoblotting analysis. Multiple labeling of trophoblast cells using immunofluorescence markers was used to investigate regional differences in localization of apoptotic cells between normal and FGR-affected fetal membranes. Apoptosis was detected in both normal and FGR-affected fetal membranes. However, quantitative analysis of apoptotic cells by immunohistochemistry showed a significant increase in FGR-affected fetal membranes compared to normal (p<0.005). Furthermore, it was observed that apoptotic cells were predominantly localized to chorio-decidual layer of the fetal membrane. By using semi-quantitative analysis of Western immunoblotting, a significant increase in the levels of the apoptotic marker proteins poly-ribo (ADP) polymerase (PARP) and the neo-epitope of cytokeratin-18 were observed in FGR-affected fetal membranes compared to normal (p<0.005). Immunofluorescence studies further confirmed the restriction of the apoptotic cells to the chorionic trophoblast cells in FGR-affected fetal membranes. Our results document for the first time an increased incidence of apoptosis in FGR-affected fetal membranes, with the apoptotic cells restricted primarily to the chorionic trophoblast layer of the fetal membranes. Increased apoptosis in FGR-affected fetal membranes may impair functions of the fetal membranes that are important for normal fetal development and growth. Elucidation of the molecular mechanisms involved in the control of apoptosis in the chorionic trophoblast layer in the FGR-affected fetal membranes may provide further insights into the etiology of FGR.

Apoptosis↗
PubMed2003

GLUT12 expression in human placenta in first trimester and term.

The aim of this study was to characterize the expression of a novel glucose transporter protein GLUT12 in human placenta. GLUT12 mRNA expression was identified by RT-PCR in extracts from five normal term placentae and in extracts from cultured cells of the JAR, JEG-3 and HTR-8Svneo cell lines. In further studies, paraffin sections of first trimester tissue from chorionic villus sampling and term tissue obtained after delivery were analysed by immunohistology with a GLUT12 specific polyclonal antibody. GLUT12 immunoreactivity was expressed predominantly in the syncytiotrophoblast and in extra-villous trophoblast cells in first trimester tissues at 10, 11 and 12 weeks' gestation. In term tissue, however, GLUT12 staining was not detected in syncytiotrophoblast and was found predominantly in villous vascular smooth muscle cells and villous stromal cells. These results suggest that there is a dynamic spatial and temporal expression pattern for the novel glucose transporter GLUT12 in human placenta.

Adult↗
PubMed2000

Determination of anterior endoderm in Xenopus embryos.

Grafts of anterior endoderm from embryos at stage 28 or later developed according to their fate (i.e., anterior) when transplanted posteriorly. Conversely, grafts from earlier embryos developed according to their new location (i.e., posterior). However, endoderm grafted along with its mesodermal and ectodermal sheath retained its fate regardless of the stage of the donor. We conclude that anterior endoderm in Xenopus embryos is determined at about stage 25 under the influence of mesoderm.

Animals↗
PubMed1999

Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif.

Induction of human plasminogen activator inhibitor type-2 (PAI-2) gene transcription is the response of macrophages to inflammatory stimuli, such as the pleiotropic cytokine, tumour necrosis factor-alpha (TNFalpha). Here we have examined whether PAI-2 gene transcription in response to TNFalpha may be mediated through a regulatory pathway involving the transcription factor, NF-kappaB. We have tested the function of two potential NF-kappaB-like sites present in the PAI-2 proximal promoter for responsiveness to TNFalpha using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFalpha regulation of the PAI-2 gene through either of these two sites, one of the NF-kappaB-like motifs, transcriptional regulatory motif (TRM), present at position -400 was found to be essential for constitutive PAI-2 transcription, as mutation of this motif abolished basal PAI-2 promoter activity in both monocyte-like U937 cells and HT1080 fibrosarcoma cells. Competition electrophoretic mobility shift assays identified four TRM-binding proteins present in U937, HT1080 and HeLa cell extracts, which bound to this motif but were not components of the NF-kappaB regulatory complex. Expression screening of a HeLa cell cDNA library using the -400 TRM as a probe identified two cDNAs encoding partial peptides which specifically bound the TRM motif. DNA sequence analysis revealed that one cDNA was novel, and the second cDNA encoded exon 5 of the nephroblastoma overexpressed (novH) proto-oncogene, suggesting a new role for this peptide in gene regulation. Taken together, these findings identify a new regulatory element required for constitutive PAI-2 transcription, and identify potential DNA-binding proteins associated with this element that may play a role in PAI-2 gene regulation.

Base Sequence↗
PubMed1999

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

Animals↗
PubMed1998

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Amino Acid Sequence↗
PubMed1998

A distal-less class homeobox gene, DLX4, is a candidate for regulating epithelial-mesenchymal cell interactions in the human placenta.

Homeobox genes of the Distal-less (Dlx) family are expressed in the vertebrate embryo in regions where epithelial cell layers contact adjacent mesenchymal cells. This study shows that the human Dlx family member, DLX4, is expressed in the placenta, primarily in regions where epithelial and mesenchymal cell layers contact. In situ hybridization studies at first trimester human placental sections revealed that DLX4 was expressed predominantly in the cytotrophoblast stem cell layer. In term placenta, DLX4 was expressed in the syncytiotrophoblast. Northern analysis revealed two DLX4 transcripts in first trimester placenta of 2.8 and 3.0 kb. Elevated levels of DLX4 mRNA were detected in a choriocarcinoma derived cell line when compared with a cytotrophoblast cell line and normal placenta. This is the first study to show that a member of the Dlx family of homeobox genes is expressed in regions of epithelial and mesenchymal cell layer contact in the human. Accumulated evidence from several studies suggest that a combinatorial code of homeobox genes is required to regulate epithelial-mesenchymal cell interactions in the vertebrate embryo. It is predicted that a similar combination of homeobox genes, that includes DLX4, is involved in regulating epithelial-mesenchymal cell interactions in extraembryonic tissues. DLX4 may also have a role in the regulation of the genes important for trophoblast invasion since the level of expression in trophoblast cell lines reflects invasive potential.

Blotting, Northern↗
PubMed1998

Reactivity of human trophoblast monoclonal antibodies with marmoset monkey trophoblast cultures.

The aim of this study was to determine whether cultured trophoblast tissues, derived from the trophectoderm of marmoset monkey blastocysts, contain homologues of human trophoblast antigens. This is an essential prerequisite to determine whether the marmoset may be a suitable model for preclinical testing of a human antitrophoblast antigen for fertility regulation. Previously evaluated monoclonal antibodies from the Flinders University laboratory, which reacted with human trophoblast with a high degree of specificity, were tested for immunohistochemical reactivity using an immunoperoxidase detection method on both frozen and paraformaldehyde-fixed sections of the cultured marmoset monkey trophoblast. All monoclonal antibodies raised against human placenta reacted positively, when compared to controls, suggesting that human and marmoset trophoblast cells share common epitopes. The specificity of the monoclonal antibodies was investigated by determining whether there was cross-reactivity with other marmoset monkey tissues, including adrenal, spleen, kidney, liver, muscle, ovary and testis. The specificities of the monoclonal antibodies on these marmoset tissues were similar to those previously found on the corresponding human tissues. We have concluded that marmoset monkey trophoblast exhibits homologues of human trophoblast antigens. The findings also suggest that marmoset monkeys should be evaluated further as a primate model to test suitable target antigens for antitrophoblast vaccines that may be useful contragestation agents in humans.

Animals↗
PubMed1998

Homeobox genes DLX4 and HB24 are expressed in regions of epithelial-mesenchymal cell interaction in the adult human endometrium.

The adult human endometrium rapidly cycles through stages of cell proliferation, differentiation and degeneration. Inappropriate endometrial cell differentiation is a contributing factor in diseases such as endometrial carcinoma and endometriosis. We have identified two homeobox genes that may play a role in the control of endometrial cell differentiation and development. In-situ mRNA hybridization experiments were used to show differential expression of DLX4 at different phases of the endometrial cycle. Higher levels of DLX4 expression were observed in proliferative phase endometrial epithelium compared with secretory phase endometrial epithelium. The HB24 homeobox gene was shown to be expressed in both the proliferative and secretory phase endometrial epithelium. We predict that DLX4 and HB24 will be required for the transcriptional control of genes important for endometrial cell differentiation. Furthermore, we propose that DLX4 and HB24 are part of a conserved combinatorial code of homeobox genes that are required for controlling epithelial-mesenchymal cell interactions in the endometrium.

Adult↗
PubMed1997

Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family, DLX4.

We have carried out a DNA binding site screen of a 32-week human placental cDNA library using a consensus homeodomain binding site as a probe. This study represents the first library screen carried out to isolate homeobox genes from the human placenta. We have shown that three homeobox genes known to be expressed in the embryo, HB24, GAX and MSX2 are also expressed in the placenta. We have also identified a novel homeobox gene, DLX4, that shows 85% sequence identity with the homeodomain encoded by the Drosophila Distal-less (Dll) gene. DLX4 therefore represents a new member of the Distal-less family of homeobox genes. This is the first evidence that members of the Distal-less family of homeobox genes are expressed in the placenta. Using fluorescence in situ hybridisation (FISH), DLX4 has been assigned to human chromosome 17q21-q22. This places DLX4 in the same region of chromosome 17 as another member of the Distal-less family, DLX3 (Scherer et al., 1995), and the HOX-B homeobox gene cluster (Acampora et al., 1989: Boncinelli et al., 1991). Members of the Distal-less family (DLX1 and DLX2; DLX5 and DLX6) are found as closely linked pairs on human chromosomes (Simeone et al., 1994). We predict that DLX3 and DLX4 are closely linked and have arisen through gene duplication and divergence from a common ancestral precursor.

Amino Acid Sequence↗
PubMed1997

Homeobox gene HB24, a regulator of haematopoiesis, is a candidate for regulating differentiation of the extra-embryonic trophoblast cell lineage.

Expression of the human homeobox gene HB24 in the cytotrophoblast stem cell population in first-trimester human placenta was investigated. HB24 expression was downregulated after cytotrophoblast had differentiated into syncytiotrophoblast. Expression of HB24 was also detected in first-trimester invasive extravillous cytotrophoblast cells. In term placenta, HB24 expression was detected in residual cytotrophoblast cells and in syncytiotrophoblast. Northern analysis revealed two HB24 transcripts in first-trimester placenta of approximately 2.5 kb and 5.3 kb. Ribonuclease protection assays were used to confirm expression of HB24 in first-trimester and term placenta and also to provide evidence that HB24 is downregulated in choriocarcinoma cell lines. In addition to being required for differentiation of the haematopoietic cell lineage, HB24 may be required for differentiation of the extra-embryonic trophoblast cell lineage.

Blotting, Northern↗