Biomedical subjects

R Fässler

Publications and source records attributed to R Fässler.

At least 109 records · Page 6Linked to original sources

PubMed1996

Differentiation and integrity of cardiac muscle cells are impaired in the absence of beta 1 integrin.

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting peacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of beta 1 integrin. Fourth, beta 1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with beta 1-null cells contain cell debris. The number of beta 1-null cells decrease from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via beta 1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.

Animals↗

PubMed1995

Transgenic mice with targeted inactivation of the Col2 alpha 1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone.

Homologous recombination in embryonic stem cells was used to prepare transgenic mice with an inactivated Col2a1 gene for collagen II, the major protein component of the extracellular matrix of cartilage. Heterozygous mice had a minimal phenotype. Homozygous mice developed into fetuses that were delivered vaginally but died either just before or shortly after birth. The cartilage in the mice consisted of highly disorganized chondrocytes with a complete lack of extracellular fibrils discernible by electron microscopy. There was no endochondrial bone or epiphyseal growth plate in long bones. However, many skeletal structures such as the cranium and ribs were normally developed and mineralized. The results demonstrate that a well-organized cartilage matrix is required as a primary tissue for development of some components of the vertebrate skeleton, but it is not essential for others.

Animals↗

PubMed1995

Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms.

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.

Amino Acid Sequence↗

PubMed1995

Structure and chromosomal localization of the mouse neurocan gene.

Cosmid clones containing the mouse neurocan gene were isolated from a genomic library using rat neurocan cDNA fragments as probe. The murine gene has a size of approximately 25 kb and contains the coding sequence for the mRNA on 15 exons. The exon-intron structure reflected the structural organization of neurocan, which is a multidomain protein belonging to the aggrecan/versican proteoglycan family. All introns between conserved modular protein domains are phase I introns. Primer extension experiments indicate a transcriptional start point 28 bases downstream of a consensus TATA sequence. Further analysis of 1 kb of 5' flanking sequence revealed in addition to AP1, AP2, and SP1 consensus binding sites multiple E-box elements and a glucocorticoid responsive element. Single-strand conformation polymorphism was used to map neurocan to chromosome 8 between the microsatellite markers D8Mit29 and D8Mit78. Among mouse mutants that have been mapped around this region are the three allelic neurological diseases tottering, leaner, and rolling. The multidomain structure and the preferential expression of neurocan in the brain suggest a potential involvement in these diseases.

Animals↗

PubMed1995

Consequences of lack of beta 1 integrin gene expression in mice.

beta 1 integrins are cell-surface receptors that mediate cell-cell and cell-matrix interactions. We have generated a null mutation in the gene for the beta 1 integrin subunit in mice and embryonic stem (ES) cells. Heterozygous mice are indistinguishable from normal littermates. Homozygous null embryos develop normally to the blastocyst stage, implant, and invade the uterine basement membrane but die shortly thereafter. Using beta 1 integrin-deficient ES cells we have established chimeric embryos and adult mice. Analysis of the chimeric embryos demonstrated the presence of beta 1 integrin-deficient cells in all germ layers indicating that beta 1-null cells can differentiate and migrate in a context of normal tissue. When evaluated at embryonic day 9.5 (E9.5), embryos with a beta 1-null cell contribution below 25% were developing normally, whereas embryos with a contribution above this threshold were distorted and showed abnormal morphogenesis. In adult chimeric mice beta 1 integrin-deficient cells failed to colonize liver and spleen but were found in all other tissues analyzed at levels from 2%-25%. Immunostaining of chimeric mice showed that in cardiac muscle, there were small, scattered patches of myocytes that were beta 1-null. In contrast, many myotubes showed some beta 1-null contribution as a result of fusion between wild-type and mutant myoblasts to form mixed myotubes. The adult chimeric brain contained beta 1-null cells in all regions analyzed. Also, tissues derived from the neural crest contained beta 1 integrin-deficient cells indicating that migration of neuronal cells as well as neural crest cells can occur in the absence of beta 1 integrins.

Animals↗

PubMed1995

Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

Animals↗

PubMed1995

Suppression of keratinocyte growth factor expression by glucocorticoids in vitro and during wound healing.

We have recently demonstrated an important function of keratinocyte growth factor (KGF) in morphogenesis of epithelium and wound re-epithelialization. Furthermore, abnormalities in KGF expression or responsiveness are associated with wound-healing defects. In this study we have analyzed the regulation of KGF expression during wound repair in glucocorticoid-treated mice that are characterized by severe wound healing abnormalities. Induction of KGF mRNA expression after skin injury was significantly reduced in these mice, whereas KGF receptor mRNA levels were only affected to a minor extent by glucocorticoid treatment. The reduced KGF expression during wound healing in steroid-treated animals is at least partially due to a direct effect of glucocorticoids on the KGF expressing mesenchymal cells, because treatment of cultured fibroblasts with dexamethasone reduced KGF mRNA levels in a time- and concentration-dependent manner. The inhibitory effect of glucocorticoids on KGF expression was compensated for by high levels of serum growth factors or pro-inflammatory cytokines, demonstrating that KGF expression is subject to positive and negative regulation. Thus it seems likely that a fine balance of various KGF-regulating factors is important for normal wound healing.

Administration, Topical↗

PubMed1995

Knockout mice: how to make them and why. The immunological approach.

Transgenic technology has developed at breakneck speed in the past years. The establishment of embryonic stem cells and the finding that they can serve as bridge between genetic manipulations in vitro and biological analysis in vivo enabled the systematic creation of mouse strains with defined genetic alterations. This review lists the strategies which can be used to alter the genetic makeup of mice and summarizes some of the results which have been obtained in genetically altered mice of immunological interest.

Animals↗

PubMed1995

Mammary epithelial cell differentiation in vitro is regulated by an interplay of EGF action and tenascin-C downregulation.

Expression of the extracellular matrix glycoprotein tenascin-C in the mammary gland is associated with cellular proliferation and cell motility during organogenesis and tumorigenesis. Because the source and the regulation of tenascin-C in these tissues are unclear, we have used tenascin-C cDNA, FITC-immunofluorescence and immuno-precipitation to examine tenascin-C expression of mammary epithelial cells. Using several mammary epithelial cell lines we could show that tenascin-C can be produced and secreted by epithelial cells. However it was found that tenascin-C synthesis was inversely correlated with the polarized epithelial phenotype. Among three mouse mammary epithelial cell clones, tenascin-C expression was most abundant in HC-11 cells, the least differentiated cell type. Expression levels were high during the growth phase but were nearly abolished when cells were grown to confluence and induced to express milk proteins. Downregulation of tenascin-C by EGF apparently commits HC-11 cells to respond to lactogenic hormones and consequently, hormone induced levels of beta-casein mRNA decreased significantly when HC-11 cells were grown on a tenascin-C substrate. On the other hand, TGF-beta, another growth factor involved in coordinated growth and differentiation of the mammary gland in vivo was found to be a very potent inducer of tenascin-C. The generation of fully polarized and tight epithelium affected the levels of tenascin-C expression. In contrast to HC-11 cells, which do not form epithelial domes in vitro, highly polarized and dome forming EpH4 and Fos-ER cells nearly lacked tenascin-C. Similarly, induction of dome formation in the rat mammary stem cell line Rama 25 by the differentiation inducer dimethylsulfoxide caused a loss of TN-C-transcripts. The inability of Fos-ER cells to develop domes in the presence of soluble tenascin-C also suggests its interference with induction and maintenance of mammary epithelial cell differentiation.

Animals↗

PubMed1994

Mice lacking alpha 1 (IX) collagen develop noninflammatory degenerative joint disease.

Type IX collagen is a nonfibrillar collagen composed of three gene products, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Type IX molecules are localized on the surface of type II-containing fibrils and consist of two arms, a long arm that is crosslinked to type II collagen and a short arm that projects into the perifibrillar space. In hyaline cartilage, the alpha 1(IX) collagen transcript encodes a polypeptide with a large N-terminal globular domain (NC4), whereas in many other tissues an alternative transcript encodes an alpha 1(IX) chain with a truncated NC4 domain. It has been proposed that type IX molecules are involved in the interaction of fibrils with each other or with other components of the extracellular matrix. To test this hypothesis, we have generated a mouse strain lacking both isoforms of the alpha 1(IX) chain. Homozygous mutant mice are viable and show no detectable abnormalities at birth but develop a severe degenerative joint disease resembling human osteoarthritis.

Animals↗

PubMed1993

Downregulation of tenascin expression by glucocorticoids in bone marrow stromal cells and in fibroblasts.

Tenascin, a predominantly mesenchymal extracellular matrix (ECM) glycoprotein has a rather restricted tissue distribution, but until now factors that inhibit its expression have not been identified. Glucocorticoids are known to be beneficial for establishment of myelopoiesis in long-term bone marrow cultures. Tenascin was found to be expressed in the bone marrow, and glucocorticoids were found to affect bone marrow tenascin expression. Both tenascin mRNAs and the mRNA of another ECM protein, laminin B1 chain, were drastically downregulated by glucocorticoids during initiation of bone marrow cultures. However, in already established long-term cultures glucocorticoids did not affect laminin B1 chain mRNA levels although tenascin mRNAs continued to be downregulated. Studies with a stromal cell line (MC3T3-G2/PA6) and fibroblasts (3T3) suggested that glucocorticoids act directly on the stromal cells that produce tenascin. In 3T3 cells this downregulation occurred within 12 h of glucocorticoid-treatment, suggesting that glucocorticoids acted through cis regulatory elements of the tenascin gene. We suggest that glucocorticoids in part regulate hematopoiesis by modifying the ECM. Furthermore, downregulation of tenascin expression by glucocorticoids may in part explain the restricted tissue distribution of tenascin in other tissues.

3T3 Cells↗

PubMed1993

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding.

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115-residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.

Amino Acid Sequence↗

PubMed1993

Expression of tenascin in developing and adult mouse lymphoid organs.

The extracellular matrix (ECM) is essential in regulating many cell functions in non-lymphoid cells, and the ECM may also play a role in the function of the immune system. Tenascin is a hexameric glycoprotein of the ECM. In mouse, two major polypeptides of MW 210 KD and 260 KD are formed by differential splicing. Northern blot screening of various mouse tissues showed that the short 6 KB tenascin message was strongly expressed in the adult thymus, whereas very little or no tenascin mRNA could be detected in spleen. In addition, immunoblotting and histological analysis with monoclonal anti-tenascin antibodies revealed the presence of tenascin in lymph nodes and spleen. In thymus, only a short-splice variant of tenascin was detected by immunoblotting, which supported the Northern blot results. Immunohistology showed that the epithelial reticular stroma in both embryonic and adult mouse thymus expressed tenascin, as did the postnatal mesenchymal reticular stroma in lymph nodes and spleen. The distribution of tenascin in the thymus was more restricted than that of fibronectin and laminin.

Animals↗

PubMed1992

An X-linked human collagen transgene escapes X inactivation in a subset of cells.

Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.

Animals↗

PubMed1990

Glucocorticoid-regulated gene expression in the immune system. Analysis of glucocorticoid-regulated transcripts from the mouse macrophage-like cell line P388D1.

To further elucidate the molecular mechanisms underlying glucocorticoid-mediated immune suppression, we have exploited cDNA cloning and subtractive screening methods to identify glucocorticoid-regulated transcripts in the mouse macrophage-like cell line, P388D1. Two of the three isolated glucocorticoid-regulated mRNA species corresponded to genes potentially important to immunoregulation: one glucocorticoid-suppressed mRNA species probably encoded the previously uncloned 3-hydroxy-3-methylglutaryl coenzyme A reductase, an enzyme that appears important for in vitro immune responses. The other mRNA species showed glucocorticoid-increased mRNA steady-state levels and was transcribed from an endogenous ecotropic type C retroviral locus. This transcript gives rise to a protein (transmembrane retroviral protein, formerly p15E), which, along with its feline and human homologs, has been implicated in immunosuppression caused by mouse, cat, and human retroviruses. Our results raise the possibility that the immunosuppressive activity of glucocorticoids might be mediated, in part, by regulating the expression of the above immunoregulatory proteins.

Animals↗

PubMed1989

Immunogenetic analysis of spontaneous autoimmune thyroiditis of obese strain chickens.

A variety of immunological, endocrinological, and virological abnormalities have been implicated in the etiopathogenesis of spontaneous autoimmune thyroiditis (SAT) of Obese strain (OS) chickens, e.g., a general T cell hyperreactivity, an increased uptake of iodine into the thyroid gland, a diminution of the glucocorticoid tonus, and an OS-specific endogenous virus. In crosses of the close-bred OS B15/B15 subline with the inbred normal CB B12/B12 strain we have studied the mode of inheritance of these aberrations and their putative association with SAT. The results indicate that none of these OS-specific characteristics alone is an absolute prerequisite for the development of thyroid infiltration, which appears to be governed by one autosomal recessive gene.

Animals↗

PubMed1989

[The behavior of hGH (human growth hormone) and somatomedin C following anesthesia induction with propofol in comparison with diazepam and thiopental].

It was the aim of present study to examine possible interactions between human growth hormone (hGH) and Somatomedin C (SmC), when inducting anaesthesia with propofol in healthy patients. Moreover these effects of propofol should be compared with that of diazepam and thiopentone. 30 patients scheduled for elective plastic-surgery were divided randomly into 3 groups. Induction of anaesthesia was performed in group 1 with diazepam, in group 2 with thiopentone and in group 3 with propofol. Measurements of hGH, SmC as well as blood glucose were performed at 4 different given times: (1.) preoperatively, (2.) 15 minutes, (3.) 30 minutes, (4.) 60 minutes after inducting anaesthesia. Diazepam and thiopentone lead to increases in hGH secretion already in a very early phase after induction of anesthesia (15 minutes) and remained on elevated level. Concentrations of Somatomedin C, as well as of blood glucose, however, remained unchanged during the whole observation period. In comparison to that findings propofol did not show any influence on hGH and consecutively on Somatomedin C activity at any time. Based on our results we can conclude that propofol should be the induction-agent of choice predominantly when anaesthetizing patients with unknown hormonal state (outpatients scheduled for operation).

Adult↗

PubMed1988

Diminished glucocorticoid tonus in obese strain (OS) chickens with spontaneous autoimmune thyroiditis: increased plasma levels of a physicochemically unaltered corticosteroid-binding globulin but normal total corticosterone plasma concentration and normal glucocorticoid receptor contents in lymphoid tissues.

Obese strain (OS) chickens afflicted with spontaneous autoimmune thyroiditis (SAT) display several signs of a general immune dysbalance, some of which may be related to altered endocrine mechanisms such as the glucocorticoid tonus. The latter is the combined result of corticosterone (CN) production, metabolism as well as excretion, and the binding of CN to corticosteroid binding globulin (CBG) and glucocorticoid receptors (GR). The present study deals with the comparative investigation of these parameters in OS and normal White Leghorn (NWL) chickens. The results obtained with radioimmunoassay for CN and radioligand saturation assays for plasma CBG as well as GR in the thymus were as follows: (1) both OS and NWL have equal total CN levels; (2) however, OS chickens exhibit elevated CBG levels, whereas the physicochemical parameters (equilibrium affinity, specificity spectrum) of CBG were equal in OS and NWL; (3) the GR capacities and affinities were equal in both OS and NWL throughout development until thymic involution. Similarly, the specificity, affinity, and sedimentation behaviour were equal in OS and NWL. (4) Furthermore, no differences were found in the response of OS and NWL splenocytes to the suppressive effect of glucocorticoids in vitro, also excluding postreceptor alterations at the cellular level in the OS. From these findings we conclude that the increased CBG levels, which are not compensated for by either increased CN plasma levels or by increased receptor capacities or affinities in lymphatic organs, represent a diminished glucocorticoid tonus in OS chickens. This may have immunoregulatory consequences which, in turn, may contribute to the development of SAT.

Animals↗