Polarization optical-histochemical characterization and supramolecular structure of carbohydrate fibrils.

Topo-optical staining reactions were used to investigate the structures of bacterial cellulose, insect chitosan and alginic acid from brown algae. Polysaccharide complexes, glycosaminoglycans and sulfate groups were presented and demonstrated selectively. Chitosan and alginic acid are structurally similar to glycosaminoglycans (GAGs), which are constituents of human amyloid fibrils. The staining sequences shown can be used as reliable methods for histochemistry with light and polarization microscopy. They will help to clarify the complex protein-polysaccharide structure of amyloid fibrils.

Inhibin/activin-betaE subunit is expressed in normal and pathological human placental tissue including chorionic carcinoma cell lines.

BACKGROUND: Inhibins and activins are important regulators of the female reproductive system. Recently, a novel inhibin subunit, named betaE, has been identified and shown to be expressed in several human tissues. However, only limited data on the expression of this novel inhibin-betaE subunit in normal and pathological human placenta as well as and human chorionic carcinoma cell lines exist. MATERIALS AND METHODS: Tissue specimens of normal, preeclamptic and HELLP pregnancies (n = 18) were obtained at the course of an cesarean section. Normal and pathological placental tissues as well as chorionic carcinoma cells (BeWo and JEG) were analyzed by using immunohistochemistry and RT-PCR. RESULTS: Expression of the inhibin betaE subunit could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by betaE-specific RT-PCR analysis. The immunoreactive score for inhibin-betaE did not show any significant differences between normal, preeclamptic and HELLP tissue in extravillous trophoblast and syncytiotrophoblast cells. Expression of inhibin betaE could further be demonstrated for the human chorionic carcinoma cell lines JEG and BeWo. DISCUSSION: We demonstrated that inhibin-betaE is expressed in normal and pathological human placenta tissues. Although the precise role of this novel inhibin subunit for human placenta development is quite unclear, similarities with the well-characterized betaA- and betaB-subunits suggest an involvement in autocrine/paracrine signaling pathways, angiogenesis, decidualization and tissue remodeling under normal as well as malignant conditions. Additionally, the human chorionic carcinoma cell lines JEG and BeWo synthesize this subunit and therefore can be used as a cell culture model for further functional analysis of this subunit in human placental tissue.

Confocal fluorescence detected linear dichroism imaging of isolated human amyloid fibrils. Role of supercoiling.

Amyloids are highly organized insoluble protein aggregates that are associated with a large variety of degenerative diseases. In this work, we investigated the anisotropic architecture of isolated human amyloid samples stained with Congo Red. This was performed by fluorescence detected linear dichroism (FDLD) imaging in a laser scanning confocal microscope that was equipped with a differential polarization attachment using high frequency modulation of the polarization state of the laser beam and a demodulation circuit. Two- and three-dimensional FDLD images of amyloids provided information on the orientation of the electric transition dipoles of the intercalated Congo Red molecules with unprecedented precision and spatial resolution. We show that, in accordance with linear dichroism imaging (Jin et al. Proc Natl Acad Sci USA 100:15294, 2003), amyloids exhibit strong anisotropy with preferential orientation of the dye molecules along the fibrils; estimations on the orientation angle, of around 45°, are given using a model calculation which takes into account the helical organization of the filaments and fibrils. Our data also show that FDLD images display large inhomogeneities, high local values with alternating signs and, in some regions, well identifiable µm-sized periodicities. These features of the anisotropic architecture are accounted for by supercoiling of helically organized amyloid fibrils.

Inhibin-betaC subunit expression in normal and pathological human placental tissues.

Inhibins and activins are important regulators of the female reproductive system. Recently, a novel inhibin betaC subunit has been identified. However, only limited data on the expression of this novel inhibin-betaC subunit in normal and pathological human placentas exist. Tissue specimens of normal, preeclamptic, hemolysis, elevated liver enzymes, low platelets (HELLP), and intrauterine growth restriction (IUGR) pregnancies (n=24) were obtained at the conclusion of a cesarean section. Normal and pathological placental tissues were analyzed by an immunohistochemical staining reaction with a specific antibody against this novel inhibin-betaC subunit. Overall, expression of the inhibin-betaC subunit could be demonstrated in normal and pathological placental tissue. The immunoreactive score (IRS) for inhibin-betaC did not show any significant differences between normal, preeclamptic, HELLP, and IUGR tissue in extravillous trophoblast and syncytiotrophoblast cells. Immunolabelling of this novel inhibin-ßC protein in normal and pathological placental tissue was demonstrated, although no differences in the staining intensity could be observed. Therefore, the inhibin-ßC isoform might not primarily be involved in the pathogenesis of these pregnancy-associated disorders. The functional role of this novel inhibin-betaC subunit in normal and pathological human placenta is still quite unclear and should thus be further investigated.

Inhibin/activin-betaC subunit in human endometrial adenocarcinomas and HEC-1a adenocarcinoma cell line.

INTRODUCTION: Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC (ßC) and betaE (ßE), have been identified. However, only limited data on the expression of the ßC subunit in human endometrioid adenocarcinomas exist. MATERIALS AND METHODS: Samples of uterine endometrioid adenocarcinomas were obtained and analysed by immunohistochemistry for the immunolabelling with an inhibin-ßC antibody. Additionally, the endometrial cancer cell line HEC-1a was used to assess the inhibin-betaC expression with the use of immunofluorescence. RESULTS: Expression of the inhibin-ßC subunit was demonstrated at the protein level by means of immunohistochemical evaluation in human endometrioid adenocarcinomas and the HEC-1a cell line. DISCUSSION: This study demonstrated, for the first time, that the novel inhibin/activin-ßC subunit is expressed in human endometrioid adenocarcinomas and in the human endometrial carcinoma cell line HEC-1a. Whether this novel ß-subunit has a substantial role in the pathogenesis and malignant transformation in human endometrium is still under investigation.

The metastasis-associated gene MTA3 is downregulated in advanced endometrioid adenocarcinomas.

The metastasis-associated gene MTA3 has an important function in invasion and metastasis of human cancer cells. Therefore, the aim of this study was to investigate the expression of this protein in endometrial adenocarcinomas and to analyse potential correlations between this nuclear transcription factor and estrogen receptors in endometrial adenocarcinomas. Additionally, we evaluated whether MTA3 might be a prognostic parameter in endometrioid adenocarcinomas. Endometrioid adenocarcinomas were obtained from 200 patients and immunohistochemically analysed for MTA3 and estrogen receptor alpha and beta (ER-alpha and ER-beta) expression. Overall, endometrioid adeno-carcinomas of histological differentiation grade 3 demonstrated a significantly lower expression of MTA3 compared to carcinomas of histological grade 1 and 2 (p<0.05). MTA3 expression is reduced in endometrioid adenocarcinomas of poor differentiation, though without any correlation to ER-alpha and ER-beta expression. Furthermore, the expression of MTA3 did not affect progression-free, cause-specific and overall survival. Overall, MTA3 did not constitute an independent prognostic factor in this study, suggesting that MTA3 is not a useful marker to assess and identify high-risk patients with endometrial adenocarcinomas. Still, the downregulation of MTA3 predispose this cell type to be of high metastatic potential after malignant transformation, playing an essential, but as yet unknown role in human endometrial carcinogenesis.

Immunohistochemical labeling of the inhibin/activin betaC subunit in normal human placental tissue and chorionic carcinoma cell lines.

Inhibins and activins are important regulators of the female reproductive system. A novel inhibin subunit, named betaC, has been identified and demonstrated to be expressed in several human tissues. We demonstrate here that inhibin betaC is expressed in human placenta. Expression of the inhibin betaC subunit was demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by an inhibin betaC-specific RT-PCR analysis. Expression of inhibin betaC was detected in the human chorionic carcinoma cell lines JEG and BeWo. Although the precise role of this novel inhibin subunit in human placenta development and homeostasis is unclear, analogies with other inhibin subunits and the strong expression of betaC in normal human trophoblast cells and chorionic carcinoma cells suggest that betaC may be involved in autocrine/paracrine signaling pathways, angiogenesis, decidualization, and tissue remodeling under normal and malignant conditions. Additionally, JEG and BeWo express betaC and, therefore, can be used as a cell culture model for further functional analysis of this subunit in the human placenta.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

BACKGROUND: Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. MATERIALS AND METHODS: Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. RESULTS: Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. DISCUSSION: Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

Peroxisome proliferator-activated receptor-gamma in normal human pregnancy and miscarriage.

Peroxisome proliferator-activated receptors (PPAR) belong to the superfamily of nuclear hormone receptors. Recent investigations emphasize a possible involvement of PPAR in obstetric and gynaecologic disorders like polycystic ovarian syndrome, endometriosis and preeclampsia. The aim of this study was to determine the frequency and distribution of peroxisome proliferator-activated receptor-gamma (PPARgamma) in normal human pregnancy and miscarriage. Placental tissue was obtained from normal human pregnancy and miscarriage during the first trimester of pregnancy. PPARgamma localisation was investigated by immunohistochemical methods. Immediate immunoreactivity of PPARgamma was observed in villous and extravillous trophoblast nuclei in normal first trimester pregnancy. A significantly enhanced labelling of PPARgamma was identified in extravillous trophoblast of miscarriage patients. This enhanced immunopositivity was also found in nuclei of villous trophoblast cells of miscarriage patients but without statistical significance. Because trophoblast invasion is negatively correlated to PPARgamma stimulation and blocking of PPARgamma leads to increased trophoblast invasion, our findings may suggest that enhanced expression of PPARgamma in abortive extravillous trophoblasts may be one factor linked to miscarriage.

The metastasis-associated genes MTA1 and MTA3 are abundantly expressed in human placenta and chorionic carcinoma cells.

Normal placenta development relies on the ability of trophoblast cells to invade into the uterus and to build up an extensively vascularized feto-maternal tissue, necessary for the nutrition of the embryo. The ability of cell migration, invasion, and the ability to induce neovascularization are likewise hallmarks of cancer cells. The metastasis-associated genes MTA1 and MTA3 are known to be involved in cancer cell migration by regulation of cell adhesion proteins and to induce the expression of neoangiogenic cytokines, as recently shown by us for ovarian cancer cells. Therefore, we analyzed the expression of MTA1 and MTA3 in normal human placenta tissues and the chorionic cancer cell lines BeWo, JEG, and JAR. Immunohistochemical analysis revealed a rather strong expression of MTA1 and MTA3 in the nuclei of human trophoblast cells. A high expression level of MTA1 and MTA3 was further observed in the nuclei of human chorionic carcinoma cells, as shown by immunofluorescence analysis, and confirmed by Western blot and RT-PCR analysis. We conclude that the high expression level of MTA proteins in human chorionic cells might facilitate trophoblast cell migration and neoangiogenesis, and might further predispose human chorionic cancer cells with properties that are characteristic for this highly aggressive and metastatic carcinoma type.

Immunohistochemical labelling of steroid receptors in normal and malignant human endometrium.

For several years it was generally believed that only a single estrogen receptor (ER) and progesterone receptor (PR) existed. However, the discovery of a new ER (ERbeta) with specificity for estrogens has induced new insights in the estrogen signalling system. Moreover, PR is expressed as two major isoforms, PR-A and PR-B that arise from alternative transcriptional starting sites within the same gene. Although PR-A and PR-B were thought to occur in similar amounts, it is now clear that they are differentially expressed and thus have distinct functions in several human tissues, including human endometrium. The ER and PR expression and distribution pattern might play an important role in normal endometrial function and pathogenesis and the expression and relationship of the two distinct ER's and PR's could be of essential clinical implications. Moreover, the imbalance in ERalpha/ERbeta expression and the PR-A/PR-B ratio might play an important role in endometrial transition and subsequently influence endometrial pathogenesis. The knowledge of the pattern of steroid receptors in human endometrial tissue is of extreme importance, since it might start a new field in hormone therapy of endometrial cancer.

Development and characterisation of an antibody for the immunohistochemical detection of inhibin/activin betaE (betaE) in normal human ovarian and placental tissue.

Inhibin/activin subunits are homologues to each other and belong to the transforming growth factor-beta (TGF-beta) family of proteins. These proteins have been demonstrated to be disulphide-linked dimers, which have a common alpha-subunit but just one of two beta-subunits, differentiated in inhibin A (alpha-betaA) and in inhibin B (alpha-betaB). Recently, an additional beta-subunit has been identified, determined as betaE and being primarily synthesized in liver tissue. However, since no antibody against the betaE subunit is commercially available, limited data on histological immunodistribution of this inhibin subunit in gynaecological organs exist. Therefore, the aims of the present study were the synthesis and evaluation of a specific antibody against the inhibin-betaE subunit. In this study, we describe the characterisation of a polyclonal antibody against the inhibin-betaE subunit. This antibody demonstrated a specific reaction in both western blot analysis and immunohistochemistry. Moreover, we demonstrated positive immunolabelling in normal human ovary and placenta. The role of this novel subunit is intriguing, especially within the view that the other inhibin/activin subunits might have substantial functions in human reproduction and carcinogenesis. However, the function of this subunit in humans remains still unclear and warrants further research.

Topo-optical reactions for the identification of O-acyl sugars in amyloid deposits.

The aldehyde bisulfite toluidine blue (ABT) reaction with former saponification (KOH-ABT) and periodic acid-borohydride reduction-saponification (PB-KOH-ABT) were applied to sections of human amyloid deposits in the respiratory tract. The saponification-induced increase in ABT-reactivity was confined to the presence of O-acyl sugars associated with the amyloid fibrils. The anisotropic and metachromatic effect in the ABT and KOH-ABT reaction was reduced in the corresponding PB-KOH-ABT reaction, a difference attributed to the removal of staining due to neutral carbohydrate residues. Since the periodic acid-borohydride reduction abolishes all pre-existing ABT-reactivity of neutral sugar vicinal diols, the isolated KOH-effect could be shown using the PB-KOH-ABT reaction. By application of this sequence, the problem identifying small quantities of O-acyl sugars was solved. It is suggested that the KOH-effect depends upon the removal of O-acyl substituents located on the polyhydroxy side chain (C7, C8, C9) of sialic acid residues. An advantage of such topo-optical reactions over biochemical techniques is the exact localization of O-acyl sugars in tissue sites. By means of the KOH-ABT and PB-KOH-ABT reactions we have demonstrated, for the first time, that O-acyl sugars occur within amyloid deposits.

Invasive hydatidiform mole: immunohistochemical labelling of inhibin/activin subunits, Ki67, p53 and glycodelin A in a rare case.

Invasive trophoblastic mole is an extremely rare condition. Its early recognition is essential since it can transform into an invasive type of tumour. Immunohistochemistry was performed with monoclonal antibodies against inhibin-alpha, -betaA and -betaB, Ki67, p53 and glycodelin A in a rare case of accidentally diagnosed invasive trophoblastic mole. There was labelling of the inhibin/activin subunits, Ki67 and p53, while glycodelin A showed minimal immunopositivity. Therefore, since the pathological diagnosis of an invasive mole is difficult, the immunohistochemical detection of inhibin/activin subunits, Ki67, p53 and glycodelin A might be additional useful tumour markers.

The relevance of the aldehyde bisulfite toluidine blue reaction and its variants in the submicroscopic carbohydrate research.

Carbohydrates are chemical compounds that contain only oxygen, hydrogen and carbon. They are classified by their number of sugar units: monosaccharides (such as glucose and fructose), and disaccharides (such as sucrose and lactose) are simple carbohydrates; oligosaccharides and polysaccharides (such as starch, glycogen and cellulose) are complex carbohydrates. Carbohydrates play a crucial role in diverse biological systems [Hricovín M. Structural aspects of carbohydrates and the relation with their biological properties. Curr Med Chem 2004;11:2565-83]. According to Roseman [Sugars of the cell membrane. In: Weissmann G, Clairborn E, editors. Cell membranes. Biochemistry, Cell Biology, Pathology. New York: H. P. Publ. Co; 1975. p. 55-64], two classes of glycoproteins are described. Free glycoproteins are localised in the surface coat of the membranes and form a thick mobile layer, without any association to the membrane itself. Functionally, however, they are located in a close association with the membrane (e.g. in the duodenal mucosa). The other group consists of the membrane glycoproteins, which are integral to the membranes and are located in the outer layer. The oligosaccharide chains are bound to the N-terminal part of proteins, and are situated in the hydrophilic zone. Glycoproteins have diverse functions. They are important in specific receptor functions, in immunological cell destruction and play a significant role in reactions with lectins, antibodies, as well as in cell association and mutual recognition of the cells. This paper focuses on aspects of a summary of polarisation optical investigations and biological functions of the following three groups of carbohydrates: oligosaccharides, glycoproteins and glycosaminoglycans.

Interaction of tomato lectin with ABC transporter in cancer cells: glycosylation confers functional conformation of P-gp.

Phospho-glycoprotein (P-gp) is a polytopic plasma membrane protein whose overexpression causes multidrug resistance (MDR) responsible for the failure of cancer chemotherapy. P-gp 170 is a member of the ATP-binding cassette (ABC) transporter superfamily and has two potentially interesting regions for drugs interfering with its efflux function, namely the oligosaccharides on the first extracellular loop with unknown function and the two intracellular ATP-binding regions providing the energy for drug efflux function. The polylactoseamine oligosaccharides on the first loop can specifically bind the tomato lectin (TL). The P-gp efflux activities of TL-pre-treated MDR resistant cells were measured in the presence of structurally unrelated resistance modifiers such as phenothiazines, terpenoids and carotenoids. The inhibition of efflux activity was measured via the increased rhodamine uptake by mouse lymphoma cells transfected in human MDR1 gene and in human brain capillary endothelial cells. The tested resistance modifiers inhibit the function of ABC transporter resulting in increased R123 accumulation in MDR1 expressing cells. TL prevented the inhibitory action of phenothiazine and verapamil on brain capillary endothelial and MDR1-lymphoma cells, presumably due to the stabilization of the functional active conformation of P-gp. Our results indicate that the polylactosamine chains of P-gp are part of the functionally active protein conformation.