Biomedical subjects

R Herken

Publications and source records attributed to R Herken.

At least 19 recordsLinked to original sources

PubMed2006

Expression of glutathione S-transferase T1 (GSTT1) in human brain tumours.

Glutathione S-transferases (GSTs) play a central role in a number of metabolic processes. Glutathione S-transferase T1 (GSTT1) is a polymorphic cytosolic enzyme and a member of the theta class of GSTs. Typical substrates for GSTT1 are industrial compounds, such as dichloromethane and ethylene oxide. It has been shown that also chemotherapeutic drugs such as BCNU [i.e. 1,3-bis(2-chloroethyl)-1-nitrosourea] are efficiently inactivated by GSTT1. BCNU is a drug which is increasingly used locally in the chemotherapy of glioblastoma multiforme WHO grade IV. Therefore, if GSTT1 were expressed in neoplastic cells of brain tumours it could be a factor for chemoresistance. In order to clarify a possible role of GSTT1 in chemoresistance, as a first step, we localized this enzyme in malignant gliomas such as glioblastoma multiforme WHO grade IV and oligodendroglioma WHO grade II. Because of its polymorphism we first genotyped the samples for GSTT1 by PCR. Using in situ hybridization, we then demonstrated that GSTT1 transcripts are expressed in neoplastic cells of both tumour types. Immunohistochemistry revealed then that whereas neoplastic cells in glioblastoma multiforme WHO grade IV contain GSTT1, it was not localized in oligodendroglioma cells. Given the polymorphism of GSTT1 and its potential activity towards BCNU, the localization of GSTT1 in glioblastoma cells can be considered as a possible factor of non-homogeneous chemotherapy response among patients with different GSTT1 genotypes.

Adult↗

PubMed2004

Ultrastructural localization of integrin subunits alpha3 and alpha6 in capillarized sinusoids of the human cirrhotic liver.

Normal liver sinusoids are not lined by a basement membrane (BM). In contrast, in the course of development of liver cirrhosis, a structured BM is formed de novo in the space of Disse. This BM contributes to the inhibition of the metabolic function of the liver but the pathogenic background of the formation of this perisinusoidal BM is still unclear. Integrins of the beta1-class are generally essential for BM stability and some of them (such as alpha2beta1, alpha3beta1 and alpha6beta1) appear de novo in the perisinusoidal space of the cirrhotic liver. Their cellular distribution in capillarized sinusoids as well as the correlation between their cellular distribution and the formation of the microvascular BM in the cirrhotic liver has not been shown at the ultrastructural level. In the present work we aimed to clarify this issue. We focused on integrins alpha3beta1 and alpha6beta1 and localised them ultrastructurally in human cirrhotic liver microvessels using postembedding immunogold which allows the ultrastructural localization of antigens with high resolution in the tissue. The newly formed basement membrane of capillarized sinusoids was visualized by means of fixation with addition of tannic acid, which enables the visualization of structures of the extracellular matrix with the highest resolution. Also, we carried out laminin detection using postembedding immunogold. Our results show that both alpha3beta1 and alpha6beta1 are expressed on the surface of both hepatocytes and endothelial cells, i.e. on both sides of the newly formed basement membrane. This latter shows zones of higher density both in close proximity to the endothelial and to the hepatocytic surfaces which resemble laminae densae. We propose that hepatocytes and endothelial cells may, therefore, by expressing such integrins, contribute to the formation of this pathological BM in the microvessels of the human cirrhotic liver. On stellate cells, which are major producers of BM components, both integrins alpha3beta1 and alpha6beta1 were also localized.

Basement Membrane↗

PubMed2001

The small proteoglycans decorin and biglycan in human articular cartilage of late-stage osteoarthritis.

OBJECTIVE: Disturbances in proteoglycan metabolism of hyaline cartilage play an essential role in the pathology of degenerative joint disease. We investigated the relation between transcript expression, protein synthesis and the ultrastructural localization of the matrix-organizing proteoglycans decorin and biglycan within intra- and extracellular compartments of late-stage osteoarthritic human articular cartilage. METHODS: Human cartilage samples of a macroscopically intact area, the adjoining area and an area of the main defect from knee joints of 10 patients with late stage osteoarthritis were investigated. In situ hybridization and immunogold histochemistry were carried out separately and in combination at the light and electron microscopic level. RESULTS: Ultrastructurally, three main chondrocyte types were identified. The highest levels of mRNA of decorin and biglycan were produced by elongated secretory type 2 cells, already known to synthesize type I collagen. Cells with high levels of mRNA also translated the corresponding proteins to be found in the extracellular compartment. The highest production rate of decorin and biglycan was seen in the tissue area adjoining the main defect. CONCLUSION: The results indicate that at late stages of osteoarthritis the levels of transcription and translation for decorin and biglycan are up-regulated, probably in an effort to compensate for the general proteoglycan loss, characteristic of this disease stage.

Aged↗

PubMed2001

Aquaporin-4 deficiency in skeletal muscle and brain of dystrophic mdx mice.

We report a detailed study of AQP4 expression in the neuromuscular system of mdx mice. Immunocytochemical analysis performed by double immunostaining revealed that mdx mice manifest a progressive reduction in AQP4 at the sarcolemmal level of skeletal muscle fast fibers and that type IIB fibers are the first to manifest this reduction in AQP4 expression. No labeling was observed in the cytoplasm of muscle fibers, indicating that the reduction in sarcolemma staining is not associated with an intracellular compartmentalization of mistargeted protein. By Western blot and RT-PCR analysis, we found that whereas the total content of AQP4 protein decreased (by 90% in adult mdx mice), mRNA levels for AQP4 remained unchanged. A similar age-related reduction in AQP4 expression was found in brain astrocytic end-feet surrounding capillaries of mdx mice. Morphometric analysis performed after immunogold electron microscopy indicated a reduction of approximately 85% in gold particles (32+/-2/microm vs. 4.7+/-0.61/microm). Western blot experiments conducted using membrane fractions from brain cortex revealed a strong reduction (of 70%) in AQP4 protein in adult mdx mice, and RT-PCR experiments demonstrated that the reduction was not at transcription level. More interesting was the finding that AQP4 reduction was associated with swelling of astrocytic perivascular processes whose ultrastructural modifications are commonly indicated as an important and early event in the development of brain edema. No apparent reduction in AQP4 was found in mdx stomach and kidney. Our data provide evidence that dystrophin deficiency in mdx mice leads to disturbances in AQP4 assembly in the plasma membrane of fast skeletal muscle fibers and brain astrocytic end-feet, suggesting that changes in the osmotic equilibrium of the neuromuscular apparatus may be involved in the pathology of muscular dystrophy.

Animals↗

PubMed2001

Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier.

In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy. In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier disruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes. All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.

Animals↗

PubMed2000

Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes.

beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of alpha 6 beta 4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiation and proliferation, and in the formation and/or maintenance of hemidesmosomes.

Animals↗

PubMed2000

Extensive glycosylation changes revealed by lectin histochemistry in morphologically normal prenatal tissues of the mouse mutant undulated (un/un).

Recently we observed that in human embryos and fetuses with a variety of malformations, not only malformed tissues, but also several non-malformed tissues displayed alterations in the glycosylation pattern. It was the aim of this work to investigate this more or less inexplicable phenomenon under experimental conditions. To this end, we studied a well known mouse model, the mouse mutant undulated, which has an exactly defined genetic defect (substitution in the pax-1 gene) leading to a localized malformation in the vertebral column. The glycosylation pattern was studied using lectin histochemistry. Distribution of binding sites for the lectins RCA I, Con A, SNA, SBA, PNA, LTA and WGA was studied during the organogenesis stages (i.e., days 11-18). It was striking that in mutants, changes in the glycosylation pattern were found not only in the malformed organ (i.e., vertebral anlage), but also in other embryonic tissues, which showed normal morphology. This suggests that the altered glycosylation seems to be a part of genetically determined phenomena throughout the entire organism. Our results show that a defect in a gene with a very restricted expression can cause universal changes in the glycosylation pattern during development.

Animals↗

PubMed2000

Interferon beta-1a prevents the effects of lipopolysaccharide on embryonic brain microvessels.

By means of light and electron microscopy we have studied the effect of interferon beta-1a (IFNbeta-1a) in the optic tecta of 20-day-old chick embryos under normal conditions and after exposure to lipopolysaccharide (LPS) which mimics the blood-brain barrier (BBB) disruption in meningoencephalitis. Optic tecta were examined for: (i) ultrastructure by means of transmission electron microscopy; (ii) the immunohistochemical localization of HT7 antigen, a specific marker of differentiation of the brain microvessels; (iii) the brain microvessel permeability, by means of horseradish peroxidase (HRP) tracer; (iv) the expression of microvessel glycoconjugates, by means of lectin histochemistry, using Ricinus communis agglutinin-I (RCA-I), specific for beta-D-galactosyl moieties and Wheat Germ agglutinin (WGA) specific for sialyl and N-acetylglucosaminyl moieties. A morphometric evaluation of brain microvessel permeability and of glycoconjugate expression was also performed. In control- and in IFNbeta-1a-treated embryos, HRP was confined to the vessel lumina which were sealed by the interendothelial tight junctions. RCA-I binding sites were recognizable both in the basal membranes and in the tight junctions, while WGA sites were present on the luminal side of the endothelial cells. HRP was blocked in the vessels lumina by the interendothelial tight junctions. After LPS treatment, HRP showed an extravascular localization and the labeling of microvessels by anti-HT7 antibodies disappeared. RCA-I binding was only found ultrastructurally and appeared as irregularly clustered gold particles, in the cleft of damaged tight junctions, but were no longer detectable in the endothelial basement membranes. After pretreatment of LPS-treated embryos with IFNbeta-1a, the vessel permeability to HRP strongly decreased and the vessels showed the normal pattern of HT7 protein and of the RCA-I binding sites. These results indicate that the changes induced by LPS in the endothelial cells are prevented by IFNbeta-1a.

Animals↗

PubMed2000

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro.

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.

Animals↗

PubMed2000

Ultrastructural colocalization of nidogen-1 and nidogen-2 with laminin-1 in murine kidney basement membranes.

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen type IV networks. Recently a new member of the nidogen family, nidogen-2, has been characterized. Preliminary immunohistochemical data indicated that nidogen-1 and nidogen-2 show a similar tissue distribution at the light microscopic level. We have now localized nidogen-1 and nidogen-2, as well as their corresponding mRNAs, at the light and electron microscopic levels in adult mouse kidney, by in situ hybridization and immunogold histochemistry, as well as carrying out double labeling with laminin-1. Both nidogen-1 and nidogen-2 mRNAs are found not only in mesenchymal cells of embryonic tissues, but also in all epithelial and endothelial cells in adult mouse kidney. Both nidogens are ubiquitous basement membrane components in the mouse kidney, being found in glomerular, tubular, and capillary compartments and Bowman's capsule. Furthermore, a substantial fraction of nidogen-1 and nidogen-2 colocalizes with laminin-1. The results indicate that nidogen-1 and nidogen-2 could well substitute for one another in some of their biological activities in kidney, for example, stabilizing basement membrane networks in vivo.

Animals↗

PubMed2000

Nidogen-1. Expression and ultrastructural localization during the onset of mesoderm formation in the early mouse embryo.

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229-237, 2000)

Animals↗

PubMed1999

Developmental expression of ZO-1 antigen in the mouse blood-brain barrier.

Tight junction biogenesis during blood-brain barrier development (BBB) in mesencephalon microvessels of mouse embryos of day 9, foetuses of day 15 and 19 and new-born (2-day-old) mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasma membrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation.

Aging↗

PubMed1999

Ultrastructural triple localization of laminin-1, nidogen-1, and collagen type IV helps elucidate basement membrane structure in vivo.

The basement membrane models which have been proposed to date are generally based on biochemical data, mainly binding studies and artificially synthesized polymers in vitro. Basically these have led to models proposing two three-dimensional laminin-1 and collagen type IV networks interconnected by nidogen-1. Whether they reflect the in vivo basement membrane structure is still not clear. We localized laminin-1, nidogen-1, and collagen type IV ultrastructurally in adult and fetal mouse kidney basement membranes with the help of immunogold-histochemistry performing double and triple localization to try to elucidate the molecular organization of basement membranes in vivo. We found laminin-1, nidogen-1, and collagen type IV distributed over the entire basement membranes in adult and fetal kidneys. This contradicts earlier studies ascribing laminin-1 to the lamina lucida and collagen type IV to the lamina densa. In addition, various basement membrane segments exhibited an organized labeling pattern for the BM components. Double-labeling revealed co-localization of laminin-1 and nidogen-1. We conclude that the combination of laminin-1 with collagen type IV as double-network basement membrane partially interconnected by nidogen-1 is found already in the early fetal kidney in vivo. However, our data cannot exclude the possibility of other variants of basement membrane assemblages. This is also indicated by a changing structure even in individual segments of one basement membrane type which renders a more flexible basement membrane architecture plausible.

Age Factors↗

PubMed1999

Immunohistochemical localization of laminin, nidogen, and type IV collagen during the early development of human liver.

There is evidence that basement membrane components control differentiation of liver sinusoids and bile ducts. These processes occur in humans in the 9th gestational week (GW). Distribution of laminin, nidogen, and type IV collagen was studied during human liver development between the 6th and the 10th GW. Laminin and nidogen lined intrahepatic microvessels in the 6th and 7th GW decreasing in quantity at the beginning of the fetal period (9th-10th GW). Type IV collagen was detected in microvessels only from the 9th GW onward. In the early periportal matrix (9th-10th GW) laminin, nidogen, and type IV collagen were diffusely distributed. At these stages, basement membrane zones of larger portal vessels and of early bile ducts were also stained for all three glycoproteins. These results show that laminin and nidogen are localized in microvessels during early human liver development and decrease in concentration at the developmental stage during which microvessels become discontinuous. In contrast, type IV collagen is not present in early microvessels but occurs when laminin and nidogen disappear. The three glycoproteins occur together only in those areas of the developing liver in which, from the 9th GW onward, the differentiation of immature liver cells into biliary epithelium takes place.

Basement Membrane↗

PubMed1999

Matrix metalloproteinases in early human liver development.

Matrix metalloproteinases (MMPs) regulate matrix deposition in tissues. Collagens I, III, and IV are involved in early human liver development. To establish whether MMPs specific for these collagens participate in early human liver development, we localized immunohistochemically MMP-1 and MMP-13 (for collagens I and III) and MMP-2 and MMP-7 (for collagen IV) in the early human liver anlage [6th-10th gestational week (GW)]. MMP-1 was found from the 6th GW onward in hepatocytes and later also in outer limiting plate hepatocytes, early bile ducts, and periportal mesenchymal cells. In the 6th GW, MMP-2 was found only in microvascular endothelium. In the 7th GW, MMP-2 was also detected in hepatocytes. From the 9th GW onward, MMP-2 was detectable in all hepatocytes and erythropoietic, endothelial, and periportal mesenchymal cells. MMP-7 was present in the 6th GW in some hepatocytes and endothelial cells, but from the 7th GW onward, only in hematopoietic cells. MMP-13 was found exclusively in hematopoietic cells. This study has shown that production of MMP -1, MMP-2, MMP-7, and MMP-13 during human liver development already occurs from the 6th GW. At this time-point their substrates are only traces or are not yet present in the tissue. A possible role of MMPs in early liver development is discussed.

Embryonic and Fetal Development↗

PubMed1999

Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma.

Immunohistochemical, cytochemical and ultrastructural data showing vivid angiogenesis and numerous mast cells (MCs) in the bone marrow of 24 patients with active multiple myeloma (MM) compared with 34 patients with non-active MM and 22 patients with monoclonal gammopathy of undetermined significance (MGUS) led us to hypothesize that angiogenesis parallels progression of MM, and that MCs participate in its induction via angiogenic factors in their secretory granules.

Adult↗

PubMed1999

Organization of the myotendinous junction is dependent on the presence of alpha7beta1 integrin.

The laminin receptor alpha7beta1 is enriched at the myotendinous junctions, and mice with a targeted inactivation of the alpha7 gene develop a form of muscular dystrophy that primarily affects this structure. By ultrastructural analysis of alpha7-deficient mice, in comparison with wild-type and mdx mice, we attempted to elucidate the role of alpha7 integrin for the integrity and function of the myotendinous junctions. Ultrastructurally, myotendinous junctions of alpha7-deficient myofibers lose their interdigitations and the myofilaments retract from the sarcolemmal membrane, whereas the lateral side of the myofibers remains morphologically normal. The basement membrane at the myotendinous junctions in alpha7 -/- mice is significantly broadened, and immunogold-histochemistry has demonstrated that the laminin alpha2 chain is not localized here but, instead, in the matrix of the neighboring tendon. In contrast, mdx mice have normal myotendinous junctions, with a matrix protein pattern also found in wild-type mice, however the lateral sides of the myofibers are severely damaged. These results suggest that the alpha7beta1 integrin is a major receptor connecting the muscle cell to the tendon and helps to organize the myotendinous junction, whereas the dystrophin-glycoprotein complex is necessary for the lateral integrity of the muscle cell.

Animals↗

PubMed1998

Comparison of lectin binding patterns in malformed and normal human embryos and fetuses.

Altered glycosylation in the course of disease detectable by changes in lectin binding patterns has been well established for adult tissues, but only a few authors have described carbohydrate entities during normal human embryonic and fetal development. Whether alterations in carbohydrate patterns occur in human embryonic and fetal tissues, affected by malformations, remains to be investigated. We, therefore, examined human embryos and fetuses at corresponding developmental stages with and without malformations (spina bifida, exencephaly, cleft lip and cleft palate, and dysmelia) with respect to their lectin binding patterns for the lectins RCA I, PNA, WGA, SBA, SNA, Con A, and LTA. Our results demonstrated that during the development of malformations, the affected tissue sites exhibited a different carbohydrate pattern from normally developed specimens. Furthermore, tissues known to be sites of secondary malformation, accompanying the primary defect, although displaying a histologically normal appearance, also showed an altered carbohydrate pattern. This might indicate a possible general alteration in the carbohydrate pattern in the course of development of malformations in man.

Binding Sites↗