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Biomedical subjects

R Herken

Publications and source records attributed to R Herken.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ultrastructural localization of microfibrillar fibulin-1 and fibulin-2 during heart development indicates a switch in molecular associations.

The microfibrillar proteins fibulin-1 and fibulin-2 were previously identified as prominent components of the endocardial cushion tissue (ECT) during heart development and shown to persist in adult valves and septa. Immunogold staining has now been used to compare their localization in embryonic (days 9-11) and adult mouse heart with that of fibronectin and the chondroitin sulphate proteoglycan versican. All four proteins were deposited in the ECT, which consists of a hyaluronan-rich, mainly unstructured matrix, but were barely detectable in myocardial basement membranes or within endocardial cells. Digestion with hyaluronate lyase selectively released the fibulins and versican but not fibronectin from the ECT. Yet neither of the two fibulins bound to hyaluronan in solid-phase assays, in contrast to versican. In the adult heart valve, all four proteins could be detected close to cross-striated collagen fibrils or microfibrils, but only versican was lost upon exposure to hyaluronate lyase. The data indicate that fibulins are associated with the hyaluronan-matrix of ECT through a bridge of versican, but that this association changes upon valve development to another supramolecular, presumably microfibrillar organization based on fibronectin and/or fibrillins.

Age Factors↗
PubMed1998

Immunohistochemical localization of glutathione S-transferase-T1 in murine kidney, liver, and lung.

Glutathione S-transferase-mediated metabolism of exogenous compounds usually leads to detoxification, but there are some exceptions. For example, glutathione S-transferase-T1 (GSTT1) can also generate genotoxic metabolites. Studies on the biology of GSTT1 are limited by the lack of specific antibodies recognizing GSTT1 in animal tissues. We localized GSTT1 immunohistochemically in mouse kidney, liver, and lung using a novel antibody targeted against the C-terminus of rat GSTT1 (rGSTT1). The antibody was characterized using immunoblot and shown to specifically recognize rGSTT1 and mouse GSTT1, but not human GSTT1. In kidney, GSTT1 staining was detected only in collecting duct epithelium. In liver, pericentral hepatocytes showed cytoplasmic and nuclear staining. Nuclear staining was also observed in several other hepatocytes without relation to liver zonation. Nuclei and supranuclear cytoplasm of bile duct epithelium and endothelium of interlobular arterioles also reacted strongly. In lung, staining was observed in bronchiolar epithelium and in surrounding muscle cells. Type II pneumocytes and endothelial cells of intrapulmonary capillaries also showed strong positive staining. This report describes the first immunohistochemical localization of GSTT1 in mammalian tissues. The reported location of GSTT1 is consistent with its known metabolic activity toward compounds such as dichloromethane and their metabolism into genotoxic products.

Animals↗
PubMed1998

Ultrastructural localization of lectin binding sites in the developing brain microvasculature.

The temporo-spatial patterning of lectin-binding sites was examined by lectin histochemistry and quantitative methods in the microvasculature of the optic tectum of 9-, 14-, 20-day-old embryos and 30-day-old chickens. Horseradish peroxidase and colloidal-gold-labelled lectins were used for detection of beta-D-galactose (RCA-I, Ricinus communis agglutinin-I) and of N-acetylglucosamine and sialic residues (WGA, Wheat germ agglutinin) at light and electron microscopical levels. At the light microscopical level, RCA-I and WGA binding sites were detectable in the early embryonic capillaries in a diffuse staining pattern; in later embryonic stages and in adult animals, RCA-I labelling became located on the abluminal surface of the vessels, while WGA staining was detected on the luminal surface. Ultrastructurally, gold labelling for RCA-I was seen intracytoplasmically in endothelial cells in 9-day-old embryos. In 14-to 20-day-old embryos and in chickens, binding sites for RCA-I were detected in endothelial tight junctions and basement membranes. In contrast, labelling of the gold-coupled WGA lectin was distributed almost exclusively on the luminal endothelial surface already in early embryos. The results indicate that the endothelial cells of the optic tectum acquire functional polarity early in their development and that glycoconjugates containing beta-D-galactose residues are involved in the biochemical composition of the tight junctions and basement membrane, which are considered to be key structures in blood-brain barrier (BBB) differentiation.

Animals↗
PubMed1998

Expression of the mouse histone gene H1t begins at premeiotic stages of spermatogenesis.

The gene encoding H1t, a testicular variant of histone H1, is expressed in mammals during spermatogenesis. Northern blot and in situ hybridization has detected H1t mRNA only at the stage of pachytene spermatocytes. We have extended this analysis to more sensitive approaches and demonstrate, by RNase protection and electron-microscopic in situ hybridization, that H1t mRNA is detectable even in spermatogonia. Just a faint H1t band is seen in Western blots of nuclear protein from 9-day-old mice. This indicates that the H1t gene is expressed at premeiotic stages, albeit at a low level. In contrast to H1t mRNA, the H1t protein has not been detected in spermatogonia by electron microscopy after immunogold staining.

Animals↗
PubMed1998

Laminin localization in enterocytic basement membrane of rat small bowel grafts. A light and electron microscopic study.

In vitro laminins stimulate numerous biological effects, such as cell migration, proliferation, attachment and differentiation. In vitro laminins influence immunocompetent cells and in vivo possibly play an important role in graft rejection. To establish how laminins could be involved in the regulation of acute rejection of small bowel allografts (with and without immunosuppression), we investigated laminin distribution in rat small bowel allografts four days after transplantation, i.e., before the onset of histological signs of rejection, using antibodies against alpha1, beta1, gamma1 chain of laminin-1. In immunosuppressed allografts, the ultrastructure of the enterocytic basement membrane appeared normal, but no laminin staining was seen in this membrane, although basement membranes of intramural blood vessels and muscle cells were normally stained. In non-operated immunosuppressed rats, laminin staining was clearly reduced in the enterocytic basement membrane, demonstrating that cyclosporin A is able to affect this membrane. Since only rats in which laminin is altered survive, this laminin alteration in the enterocytic basement membrane presumably plays an important role in overcoming the acute rejection.

Animals↗
PubMed1998

Light and electron microscopic in-situ hybridization of collagen type I and type II mRNA in the fibrocartilaginous tissue of late-stage osteoarthritis.

OBJECTIVE: Biochemical analysis indicates the presence of collagen type I in fibrocartilaginous tissue of osteoarthritic cartilage, whereas normal hyaline cartilage contains only collagen type II produced by normal chondrocytes. Fibrocartilaginous tissue of late-stage osteoarthritis also exhibits irregularly shaped type 2b secretory chondrocytes as described in the literature. We have attempted to elucidate the type of cell which produces each type of collagen in late-stage osteoarthritis. DESIGN: We carried out in-situ hybridization at the light and electron microscopic level on the same tissue embedded in LR-Gold applying silver enhancement for gold-coupled anti-DIG antibodies. We correlated the types of cells with the expression of transcripts for type I and type II collagen. RESULTS: We found that cells resembling type 2b secretory chondrocytes of deep zones of fibrocartilaginous tissue expressed collagen type I mRNA and almost no collagen type II mRNA. The amount of collagen type I mRNA was as high as the amount produced in normal human skin fibroblasts. CONCLUSION: Some of the collagen type I in osteoarthritic human cartilage of late-stage disease is produced by cells resembling type 2b secretory chondrocytes of the deep zone.

Adult↗
PubMed1998

Free laminin in the extracellular matrix of B-cell non Hodgkin's lymphomas.

The authors show that in the perivascular stroma of B-cell non-Hodgkin's lymphomas (B-NHL) is present a granular, speckled pattern of expression of laminin, similar to the 'free-laminin' firstly described by Lugassy et al. (1997) in human melanoma. The role of this form of laminin may be to promote the migration of tumor cells, which express a variety of binding-laminin surface proteins, whose expression is significantly increased in several human cancer models.

Extracellular Matrix↗
PubMed1997

Changes in lectin binding sites during early human liver development.

In this study we investigated whether changes in glycosylation during liver morphogenesis correlate with the early development of individual structures in the human liver. Therefore, we localized the binding of the lectins from Sambucus nigra (SNA; specific for sialic acid), Triticum vulgare (WGA; specific for N-acetylglucosamine and sialic acid), Ricinus communis (RCA I; specific for beta-galactose), Lotus tetragonolobus (LTA; specific for alpha-fucose) and Concanavalia ensiformis (Con A; specific for alpha-mannose) in the human liver between the 4th and the 12th gestational week (GW). Cell membranes of early hepatocytes (5th-6th GW) showed strong staining for RCA I, which decreased noticeably from the 8th-9th GW onward. Early intrahepatic capillaries (4th-5th GW) showed reactions only for WGA and RCA I. Reactions for SNA occurred later (6th-9th GW). At this time a fine granular staining for SNA was visible at the sinusoidal sides of hepatocytes. The hepatocytes of the outer limiting plate were specifically stained by WGA, Con A, and SNA in the 9th GW and the staining remained visible in developing bile ducts up to the 12th GW. The possible biological significance of the appearance or disappearance of carbohydrate moieties during early human liver development is discussed.

Binding Sites↗
PubMed1997

The oncoproteins c-erb-B2, c-fos and the tumour suppressor protein p53 in human embryos and fetuses.

Oncoproteins and tumour-suppressor proteins are thought to possess an antagonistic function in the regulation of growth and differentiation processes during embryonic and fetal development. In contrast, in the adult, tumour growth is associated with the overexpression of oncoproteins or the malfunction of tumour-suppressor proteins. We examined the occurrence of the tumour proteins c-erb-B2 and c-fos and the tumour-suppressor protein p53 in 17 human embryos and fetuses with the help of immunohistochemistry. C-erb-B2 was detected mainly in embryonic tissue that are not known for c-erb-B2-overexpression in tumours in the adult. In contrast, c-fos was almost always located in fetal tissues corresponding to its location in adult tumours. Staining for p53 was found in a wide variety of embryonic and fetal tissues. C-erb-B2 and p53 were localized in the same tissue structures of the developing skin, heart and muscle. In other tissues, e.g. muscle and bone, c-fos was found together with p53, suggesting an antagonistic action of these proliferative and antiproliferative factors. Furthermore, c-erb-B2. c-fos and p53 appear to be important for growth and differentiation processes in human development as the occurrence of these proteins was not only restricted to specific tissues but also to specific stages of development of these tissues.

Embryo, Mammalian↗
PubMed1997

Immunohistochemical localization of the small proteoglycans decorin and biglycan in human intervertebral discs.

Immunohistochemistry was used to study the presence and distribution of the core proteins of the small proteoglycans decorin and biglycan in the various compartments of human intervertebral discs. Both proteoglycans could be found in the outer tendon-like parts of the annulus fibrosus, indicating their potential role in collagen network formation and biomechanical stress resistance. The loss of both proteoglycans in the annulus of individuals older than 50 years reflects a normal age-related change. In the nucleus pulposus, decorin could be found in fibrillar areas of the interterritorial matrix, thereby indicating co-localization of decorin with fibrils containing type II collagen. Biglycan was present in the extracellular matrix of the nucleus pulposus of adults. The pericellular immunoreactive rims observed around nucleus pulposus cells and giant chondrones indicated local biosynthetic activity for these small proteoglycans. The staining patterns in cartilage endplates resembled those found in human hyaline articular cartilage.

Adolescent↗
PubMed1997

Ultrastructural localization of binding sites for the lectins RCA I, WGA, and LTA in the preimplantation mouse embryo.

In biological tissues, specific carbohydrate moieties of the oligosaccharide chains of glycoproteins can be localized by lectin binding. Such carbohydrate moieties are among the factors that mediate cell-cell or cell-matrix interactions during pre- and postimplantation embryonic development. Binding sites for the lectins RCA I, WGA, and LTA were localized in preimplantation mouse embryos at the ultrastructural level with the help of postembedding lectin gold cytochemistry. WGA and RCA I binding sites, but no LTA binding sites, were present in the zona pellucida. WGA and RCA I binding sites were found at cell surfaces of morulae and in cells of the inner cell mass of blastocysts, suggesting that N-acetylglucosamine-, terminal beta-galactosyl-, and N-acetylgalactosamine-rich glycoproteins might be involved in cell-cell and cell-matrix interactions. WGA binding sites were found predominantly in electron lucid vesicles of the blastomeres, whereas RCA I was detected in electron dense vesicles of the compacted morula and later in the polar trophoblast cells. This allows early identification of blastomere cells that later differentiate into the polar trophoblast.

Animals↗
PubMed1996

Localization of large-T oncoprotein during the embryonic and fetal development of transgenic mice.

Oncoproteins are not only involved in the development of tumors but also play a role in physiological regulation in embryonic growth and differentiation. The mechanisms by which regulation is accomplished in embryonic stages differ from postnatal or adult stages. Oncoproteins responsible for tumors in the adult, i.e., products of proto-oncogenes, are prevented from causing tumors in the embryo. If oncogenes are introduced artificially into the embryo. will they be governed by the embryonic regulation described above? To answer this question we used transgenic mice in which the hybrid construct MSV-SV40-Large-T, composed of the Simian-Virus-Oncogene. Large-T with its SV40-promotor and the Moloney Murine Sarcoma Virus (MSV)-enhancer, had been integrated. Under the influence of large-T expression, these animals develop either brain or endocrine pancreas tumors. In the present investigation, we localised large-T expression during development of mouse embryos and fetuses. Interestingly, we saw large-T positive reactions in organ anlagen other than those that later develop tumors. We found large-T antigens in cartilage anlagen, e.g., in ribs and vertebrae, particularly in fetuses of days 14 to 17, and also in a variety of epithelial cells such as in the lung or the choroid plexus. Our results indicate that, as for proto-oncogene products, the effect of an artificially introduced transgenic oncogene product can also be regulated by embryonic cells.

Animals↗
PubMed1996

Expression pattern of type II collagen mRNA during early vertebral development in the human embryo.

The role of extracellular matrix molecules in ontogenetic differentiation processes is a matter of increasing interest. In cartilage development, type II collagen is suspected of promoting chondrogenic differentiation, since its expression has been demonstrated in a range of precartilaginous tissues of vertebrate species. Up to now, no studies supplying a coherent description of type II collagen expression in the skeletogenesis of human embryos including early embryonic stages have been published. In this work, we examine the temporal and spatial distribution of type II collagen mRNA during vertebral column development in human embryos from 4 to 12 weeks of gestation using non-radioactive in situ hybridization. The onset of gene expression was demonstrable in the 5th week in precartilaginous mesenchymal cells and in notochordal cells. Additionally, we found a weaker hybridization signal in the mesenchymal precursors of the intervertebral discs. In the anlagen of the axial skeleton, type II collagen expression decreased during osteogenic reconstruction in the 11th/12th week, whereas expression continued in the notochordal remnants of the future nuclei pulposi. The results suggest a relatively late occurrence of type II collagen in human vertebral development compared with other vertebrate species. The distribution of gene expression is concordant with a possible role of this molecule in promoting differentiation of mesenchymal cells into chondrocytes. The mechanism of this influence remains to be established.

Cartilage↗
PubMed1996

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo.

Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4-10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvements in the differentiation of heart, skeletal and neuronal structures.

Animals↗
PubMed1996

Glycosaminoglycans modulate cell-matrix interactions of human fibroblasts and endothelial cells in vitro.

Contact of various cells with extracellular matrix molecules modulates their cellular functions and phenotype. Most investigations have employed dishes coated with purified matrix constituents or plain collagen I lattices omitting the effects of other important matrix components such as proteoglycans. In this study we analyze the effect of purified glycosaminoglycans (GAGs) on human fibroblasts and human umbilical vein endothelial cells (HUVEC) embedded within collagen I/III lattices. HUVEC contracted collagen I/III gels far less efficiently than fibroblasts and addition of heparan sulfate and heparin almost completely inhibited contraction. In collagen gels HUVEC down-regulated collagenase mRNA while increasing collagen I, IV mRNA expression. Addition of heparin and heparan sulfate reversed the collagen IV mRNA induction whereas hyaluronic acid and chondroitin sulfate enhanced fibronectin and collagenase transcripts. Fibroblasts readily contracted collagen gels, and mRNA levels for fibronectin, collagenase and interleukin-6 were stimulated. Gel contraction was mostly unaffected by the different glycosaminoglycans. Fibroblasts responded to the addition of dermatan sulfate, heparan sulfate and heparin with a decrease in fibronectin, collagenase and interleukin-6 mRNA. Binding studies revealed saturable binding sites on fibroblasts and HUVEC for 35S-labelled heparin, demonstrating specificity for heparin and heparan sulfate over other GAGs in competition experiments. This study implies that glycosaminoglycans participate in cell-matrix interactions by effectively modulating the cellular phenotype via high affinity binding sites.

Animals↗
PubMed1995

Development of Reichert's membrane in the early mouse embryo.

Although the composition of Reichert's membrane, a thick multilayered basement membrane between the parietal endoderm cells and the trophoblast cells of rodents, has often been investigated, the site of its production remains a subject of controversial discussion. In particular, the role of the trophoblast cells is unclear. In the present work we examined the initial development of Reichert's membrane in the early mouse embryo, using glutaraldehyde fixation with tannic acid. In the early blastocyst the occurrence of a tannic-acid-positive layer located at the inner surface of the mural trophoblast indicated the onset of basement membrane formation by the trophoblast cells. In the peri-implantation phase, this basement membrane extended into lateral areas of the inner cell mass separating the newly differentiated ectoderm and endoderm cells from each other. In these lateral regions, where the recently formed primitive endoderm cells had been attached to the monolayered basement membrane of the mural trophoblast cells, followed by an apposition of basement membrane material, probably synthesized by primitive endoderm cells, along this primary membrane.

Animals↗
PubMed1995

Intermediate filament typing of the human embryonic and fetal notochord.

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and 19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.

Antibodies, Monoclonal↗