Biomedical subjects

A Aszódi

Publications and source records attributed to A Aszódi.

At least 19 recordsLinked to original sources

PubMed2000

Structure and mapping of the mouse matrilin-3 gene (Matn3), a member of a gene family containing a U12-type AT-AC intron.

The gene for murine matrilin-3, an extracellular matrix protein present in cartilage, was isolated and further characterized. The gene spans 23.4 kb and comprises 8 exons; with one exception, this reflects the modular structure of the protein. The major and a minor transcription start site were determined by RNase protection assays to positions approximately 72 nt and 87 nt upstream of the ATG codon, respectively. The promoter contains a TATA-like box 32 bp upstream of the main transcription start as well as several potential binding sites for eukaryotic transcription factors. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, does not follow the GT-AG rule and belongs to the subgroup of introns having AT-AC at the ends that are spliced by the U12-type spliceosome. The mouse matrilin-3 gene does not contain hidden exon sequences coding for the second vWFA-like domain present in all other matrilins. The intron that could possibly contain such sequences instead shows 75% repetitive sequences, indicating an evolutionary process that has led to the loss of sequences coding for vWFA2. Single-strand conformation polymorphism analysis was used to map the Matn3 gene to the proximal end of Chr 12, linked to the genes Synd1, Apob, Dntb, and Kif3c.

Amino Acid Sequence↗

PubMed2000

High-throughput functional annotation of novel gene products using document clustering.

Gene products differentially expressed in healthy vs. diseased tissues may be considered drug targets since the change in their expression level can be related to the cause and progression of the disease studied. A significant portion of the proteins produced by these genes will be unknown and consequently their function must be characterised. The experimental elucidation of biochemical function must be supported by computational tools which can help predicting the possible function of a given protein from its amino acid sequence. We have designed a high-throughput system which automatically analyses amino acid sequences deduced from differentially represented cDNA clones. The system attempts to assign a biological function to protein sequences by carrying out searches in sequence databanks and by locating functionally relevant motifs in the query sequences. The results delivered by the various prediction methods consist of the annotations of matching sequences and/or motifs, which are free-format texts written by humans and therefore may describe the same concept with synonymous words. It is desirable to present the results in such a way that the annotations describing the same biological function are grouped together. To this end we devised an algorithm that enables the hierarchical clustering of free-format documents based on their contents. The system is capable of detecting and flagging conflicting annotations, and will speed up the interpretation of the function prediction results.

Algorithms↗

PubMed2000

Mammalian skeletogenesis and extracellular matrix: what can we learn from knockout mice?

Formation of the vertebrate skeleton and the proper functions of bony and cartilaginous elements are determined by extracellular, cell surface and intracellular molecules. Genetic and biochemical analyses of human heritable skeletal disorders as well as the generation of knockout mice provide useful tools to identify the key players of mammalian skeletogenesis. This review summarises our recent work with transgenic animals carrying ablated genes for cartilage extracellular matrix proteins. Some of these mice exhibit a lethal phenotype associated with severe skeletal defects (type II collagen-null, perlecan-null), whereas others show mild (type IX collagen-null) or no skeletal abnormalities (matrilin-1-null, fibromodulin-null, tenascin-C-null). The appropriate human genetic disorders are discussed and contrasted with the knockout mice phenotypes.

Animals↗

PubMed1999

Perlecan maintains the integrity of cartilage and some basement membranes.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Animals↗

PubMed1999

Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.

Fibromodulin is a member of a family of connective tissue glycoproteins/proteoglycans containing leucine-rich repeat motifs. Several members of this gene family bind to fibrillar collagens and are believed to function in the assembly of the collagen network in connective tissues. Here we show that mice lacking a functional fibromodulin gene exhibit an altered morphological phenotype in tail tendon with fewer and abnormal collagen fiber bundles. In fibromodulin-null animals virtually all collagen fiber bundles are disorganized and have an abnormal morphology. Also 10-20% of the bundles in heterozygous mice are similar to the abnormal bundles in fibromodulin-null tail tendon. Ultrastructural analysis of Achilles tendon from fibromodulin-null mice show collagen fibrils with irregular and rough outlines in cross-section. Morphometric analysis show that fibromodulin-null mice have on the average thinner fibrils than wild type animals as a result of a larger preponderance of very thin fibrils in an overall similar range of fibril diameters. Protein and RNA analyses show an approximately 4-fold increase in the content of lumican in fibromodulin-null as compared with wild type tail tendon, despite a decrease in lumican mRNA. These results demonstrate a role for fibromodulin in collagen fibrillogenesis and suggest that the orchestrated action of several leucine-rich repeat glycoproteins/proteoglycans influence the architecture of collagen matrices.

Animals↗

PubMed1999

The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function.

The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP- and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP-deficient mice had normal agonist-induced contraction, and normal cAMP- and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP- and cGMP-mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP- and cGMP-dependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.

Animals↗

PubMed1999

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Animals↗

PubMed1998

Collagen II is essential for the removal of the notochord and the formation of intervertebral discs.

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.

Animals↗

PubMed1998

Endochondral ossification is dependent on the mechanical properties of cartilage tissue and on intracellular signals in chondrocytes.

Skeletal elements are formed either by replacing a performed cartilagenous matrix template in a process called endochondral ossification or directly from mesenchyme by a process known as membranous ossification. Longitudinal growth of bones is achieved by growth plates where calcified cartilage is converted into bone. To investigate the role of extracellular matrix as well as intracellular signaling pathways in the formation and growth of bone, the genes coding for type II collagen and cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGK) II, were disrupted. It is demonstrated that loss of Col2a1 or cGKII led to abnormal endochondral ossification and skeletal development. In cGKII -/- mice, bones derived by membranous ossification developed normally while bones derived by endochondral ossification were shortened. This growth defect was not associated with a general metabolic disturbance. In Col2a1 knockout mice, endochondral ossification was completely absent, whereas membraneous ossification was not affected. Despite the defects in bone formation, invasion of blood vessels into bone cavities and formation of bone marrow occurred in Col2a1-null mice. Taken together, the phenotypes of these two knockout mice show that chondrocytes need a well-functioning extracellular matrix scaffold and a normal cGMP-signaling system for endochondral ossification to form a normal skeleton.

Animals↗

PubMed1998

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

Animals↗

PubMed1998

Mouse models for extracellular matrix diseases.

Mutations of a number of genes encoding for extracellular matrix (ECM) proteins in mice have provided new insights regarding their role during development and disease. Many mouse strains have helped to verify the link between mutation and disease in humans, and others have produced unexpected phenotypes and identified new functions for ECM proteins. Finally, some null mutations in ECM genes provide no phenotypic alterations in mice, confronting the scientific community with a new challenge to search for their functions. This review lists all mouse strains with spontaneous and experimentally induced mutations in ECM genes. The phenotypes of these mice are discussed in comparison with the human diseases.

Animals↗

PubMed1998

Sequence, structure and chromosomal localization of Crtm gene encoding mouse cartilage matrix protein and its exclusion as a candidate for murine achondroplasia.

The mouse cartilage matrix protein gene (Crtm) was isolated from a cosmid library using a mouse Crtm cDNA fragment as probe. Crtm spans 12.2 kb from the start of translation to the polyadenylation signal sequence and comprises eight exons. Sequencing of the 1.9 kb 5' flanking region revealed a TATA-like box 72 bp upstream from the initiator Met codon as well as several cis-acting motifs known to bind eukaryotic transcription factors. Analysis of the exon-intron junctions demonstrated that the last intron does not follow the gt/ag rule but belongs to the minor class of pre-mRNA introns that contain "at" and "ac" at their 5'and 3' ends, respectively. Single-strand conformation polymorphism analysis was used to map Crtm to the distal part of chromosome 4 between the microsatellite markers D4Mit16 and D4Mit339. Achodroplasia (cn), a recessive skeletal disorder in mice, has already been mapped to this region. Immunostaining for CMP and sequence of Crtm in cn/cn mice failed to reveal any disease-specific mutations, suggesting that mutations in Crtm do not cause achondroplasia.

Achondroplasia↗

PubMed1998

Regulation of cell-type specific expression of lacZ by the 5'-flanking region of mouse GAD67 gene in the central nervous system of transgenic mice.

The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by beta-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5'-upstream regulatory region of GAD67 and E. coli lacZ. No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers. In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites. The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5'-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region. Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects. Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system.

Animals↗

PubMed1997

Absence of the alpha1(IX) chain leads to a functional knock-out of the entire collagen IX protein in mice.

Cartilage fibrils contain collagen II as well as smaller amounts of collagens IX and XI. The three collagens are thought to co-assemble into cartilage-specific arrays. The precise role of collagen IX in cartilage has been addressed previously by generating mice harboring an inactivated Col9a1 gene encoding the alpha1(IX) chain, i.e. one of the three constituent chains of collagen IX (Fässler, R., Schnegelsberg, P. N. J., Dausman, J., Shinya, T., Muragaki, Y., McCarthy, M. T., Olsen, B. R., and Jaenisch, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5070-5074). The animals did not produce alpha1(IX) mRNA or polypeptides and were born with no conspicuous skeletal abnormality but post-natally developed early onset osteoarthritis. Here we show that the deficiency in alpha1(IX) chains leads to a functional knock-out of all polypeptides of collagen IX, whereas the Col9a2 and Col9a3 genes were normally transcribed. Therefore, synthesis of alpha1(IX) polypeptides is essential for the assembly of heterotrimeric collagen IX molecules. Surprisingly, cartilage fibrils of all shapes and banding patterns found in normal newborn, adolescent, or adult mice were formed in transgenic animals, although they lacked collagen IX. Therefore, collagen IX is not essential, and may be functionally redundant, in fibrillogenesis in cartilage in vivo. The protein is required, however, for long term tissue stability, presumably by mediating interactions between fibrillar and extrafibrillar macromolecules.

Animals↗

PubMed1997

Protein modeling by multiple sequence threading and distance geometry.

The application of homology modeling is often limited by the lack of known structures with sufficiently high sequence similarity to the target protein. The recent development of threading methods now enable the identification of likely folding patterns in a number of cases where the structural relatedness between target and template(s) is not detectable at the sequence level. We devised a hybrid method in which fold recognition was performed using the Multiple Sequence Threading (MST) method. The structural equivalences deduced from the threading output were used to guide the distance geometry program DRAGON in the construction of low-resolution C alpha/C beta models. The initial structures were converted to full-atom representation and refined using the general-purpose molecular modeling package QUANTA. The performance of the approach is illustrated on the CASP2 target T0004 (polyribonucleotide nucleotidyl-transferase S1 motif (PNS1) from Escherichia coli, PDB code: 1SRO) for which no obvious homologues with known structure were available. The correct fold of PNS1 was successfully identified, and the model was found to be more similar to the experimental PNS1 structure than the scaffold (C alpha RMSD of 6.2 A compared with 6.4 A). Our results indicate that a sensitive fold recognition algorithm coupled with a distance geometry program capable of rapidly generating initial structures can successfully complement high-resolution homology modeling methods in cases where sequential similarity is low.

Escherichia coli↗

PubMed1997

Distance geometry based comparative modelling.

A distance geometry based protein modelling algorithm is presented which relies on the projection of simple model chain coordinates into Euclidean spaces with gradually decreasing dimensionality. Fast embedding was achieved by performing separate distance matrix projections on subsets of the model points. Structural equivalences between the unknown target and related proteins with known structures were deduced either from a mixed sequence/structure multiple alignment or from the output of various fold recognition (threading) approaches. These equivalences were mapped onto the model as structure-specific conserved C alpha atom distances and secondary structure assignments. Additional nonspecific distance restraints derived from general stereochemical properties of folded protein chains were used to guide the modelling process. The method quickly constructed a large number of low-resolution models which could then serve as starting conformations for full-atom refinement. Structure predictions for some targets in the 'Asilomar Challenge' (CASP2) are presented to illustrate potential applications of the approach.

Algorithms↗

PubMed1996

Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II.

Cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP. Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates. These results indicate that cGKII plays a central role in diverse physiological processes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Cloning, sequencing and expression analysis of mouse cartilage matrix protein cDNA.

A cDNA encoding the mouse cartilage matrix protein (CMP) was cloned following the reverse-transcription polymerase chain reaction and rapid amplification of cDNA ends procedures using mRNA isolated from trachea. The open reading frame encodes a product of 500 amino acids. Large parts of the protein have been completely conserved when compared to chicken and human sequences, including all 12 cysteine residues of the mature CMP. In situ hybridization reveals an even distribution of the CMP mRNA in the developing skeleton, which is followed by a zonal distribution paralleling hypertrophy and calcification. From early cartilage differentiation and onwards, CMP transcript is absent in the forming articular surfaces and intervertebral discs. Extraskeletal expression of CMP mRNA was detected in the adult eye.

Amino Acid Sequence↗