Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.     

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.     

Fair Electronic Payment Scheme Based on DSA

We present a multi-signature scheme based on DSA and describes a fair electronic payment scheme based on improved DSA signatures. The scheme makes both sides in equal positions during the course of electronic transaction, A Trusted Third Party (TTP) is involved in the scheme to guarantee the fairness of the scheme for both sides. However, only during the course of registration and dispute resolution will TTP be needed. TTP is not needed during the normal payment stage.     

A histone H3 methyltransferase controls DNA methylation in Neurospora crassa.

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. We have demonstrated previously that dim-2 encodes a DNA methyltransferase that is responsible for all known cytosine methylation in Neurospora crassa. Here we report that another Neurospora gene, dim-5, is required for DNA methylation, as well as for normal growth and full fertility. We mapped dim-5 and identified it by transformation with a candidate gene. The mutant has a nonsense mutation in a SET domain of a gene related to histone methyltransferases that are involved in heterochromatin formation in other organisms. Transformation of a wild-type strain with a segment of dim-5 reactivated a silenced hph gene, apparently by 'quelling' of dim-5. We demonstrate that recombinant DIM-5 protein specifically methylates histone H3 and that replacement of lysine 9 in histone H3 with either a leucine or an arginine phenocopies the dim-5 mutation. We conclude that DNA methylation depends on histone methylation.     

A region of the Drosophila genome necessary for CNS development.

Mutations in genes involved in essential aspects of central nervous system development in Drosophila melanogaster are expected to be lethal. Thus, when searching for neurogenic mutants attention should be focused on embryonic lethal point mutants, for many of these might affect neural development. However, this approach can be very time consuming, for the location of neurogenic genes is unknown. A more convenient approach, which allows a faster screening of the genome, is to use relatively small chromosome deletions to determine whether the lack of a definite part of the genome affects neurogenesis. Once any region producing an interesting neural phenotype is found, it can be further analysed by the use of smaller deletions or point lethal mutants mapping within it, until the gene(s) responsible can be more precisely localised. We report here on a region of the Drosophila genome which has been found necessary for normal neurogenesis.     

Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer

Recently, we identified recurrent gene fusions involving the 5' untranslated region of the androgen-regulated gene TMPRSS2 and the ETS (E26 transformation-specific) family genes ERG, ETV1 or ETV4 in most prostate cancers. Whereas TMPRSS2-ERG fusions are predominant, fewer TMPRSS2-ETV1 cases have been identified than expected on the basis of the frequency of high (outlier) expression of ETV1 (refs 3-13). Here we explore the mechanism of ETV1 outlier expression in human prostate tumours and prostate cancer cell lines. We identified previously unknown 5' fusion partners in prostate tumours with ETV1 outlier expression, including untranslated regions from a prostate-specific androgen-induced gene (SLC45A3) and an endogenous retroviral element (HERV-K_22q11.23), a prostate-specific androgen-repressed gene (C15orf21), and a strongly expressed housekeeping gene (HNRPA2B1). To study aberrant activation of ETV1, we identified two prostate cancer cell lines, LNCaP and MDA-PCa 2B, that had ETV1 outlier expression. Through distinct mechanisms, the entire ETV1 locus (7p21) is rearranged to a 1.5-megabase prostate-specific region at 14q13.3-14q21.1 in both LNCaP cells (cryptic insertion) and MDA-PCa 2B cells (balanced translocation). Because the common factor of these rearrangements is aberrant ETV1 overexpression, we recapitulated this event in vitro and in vivo, demonstrating that ETV1 overexpression in benign prostate cells and in the mouse prostate confers neoplastic phenotypes. Identification of distinct classes of ETS gene rearrangements demonstrates that dormant oncogenes can be activated in prostate cancer by juxtaposition to tissue-specific or ubiquitously active genomic loci. Subversion of active genomic regulatory elements may serve as a more generalized mechanism for carcinoma development. Furthermore, the identification of androgen-repressed and insensitive 5' fusion partners may have implications for the anti-androgen treatment of advanced prostate cancer.     

Clinical and genetic analysis of two Chinese families with benign familial neonatal convulsions

Benignfamilialneonatalconvulsions(BFNC)is arareautosomaldominantinheritedepilepsysyn dromecharacterizedbyunprovokedpartialorgeneral izedseizures.Theseizuresusuallyoccurfromthesec onddayofbirthtothesixthmonthandremitsponta neouslyafterseveralweekstomonths.Mostindivid ualsareseizure freebytheageofsixmonths.The serumchemistryandneuroradiologicalexaminations,interictalelectroencephalogram(EEG),andpsy chomotordevelopmentareusuallynormal.However,10%to15%ofpatientshavetheriskofseizurere currencela…     

Clinical and genetic analysis of two Chinese families with benign familial neonatal convulsions

Benign familial neonatal convulsions (BFNC) is a rare autosomal dominant inherited epilepsy syndrome. Two voltage-gated potassium channel genes, KCNQ2 and KCNQ3, have been identified as the genes responsible for BFNC. Here we report two Chinese families with clinical histories of typical BFNC. Using six microsatellite markers, two located at KCNQ2 locus and four at KCNQ3 locus, linkage analysis was performed in the two families, which excluded the linkage of BFNC to KCNQ3, but could not exclude the linkage to KCNQ2. Direct DNA sequencing of the KCNQ2 gene in the two families was performed, and two formerly unknown polymorphisms were identified, but no KCNQ2 mutation was found in the two families. Our study suggests the genetic heterogeneity in Chinese families with BFNC and proves the existence of a new gene locus for BFNC.     

Embryological and molecular investigations of parental imprinting on mouse chromosome 7

Mouse embryos with duplications of whole maternal (parthenogenetic and gynogenetic) or paternal (androgenetic) genomes show reciprocal phenotypes and do not develop to term. Genetic complementation has identified the distal region of chromosome 7 (Chr 7) as one of the regions for which both a maternal and paternal chromosome copy are essential for normal development, presumably because of the presence of imprinted genes whose expression is dependent on their parental origin. Embryos with the maternal duplication and paternal deficiency of distal Chr 7 are growth retarded and die around day 16 of gestation; the reciprocal paternal duplication embryos die at an unidentified earlier stage. We report here the incorporation of cells with the paternal duplication into chimaeras, resulting in a striking growth enhancement of the embryos. One gene located on mouse distal Chr 7 (ref. 5) is the insulin-like growth factor 2 (Igf2) gene, an embryonic mitogen. In embryos with the maternal duplication of distal Chr 7, the two maternal alleles of the Igf2 gene are repressed. The presence of two paternal alleles of this gene in many cells is probably responsible for the growth enhancement observed in chimaeras. We propose that there are other imprinted genes in this Chr 7 region. We also compare the imprinting of this subgenomic region with phenotypes resulting from the duplication of the whole parental genome in parthenogenones and androgenones.     

Shank3 mutant mice display autistic-like behaviours and striatal dysfunction

Autism spectrum disorders (ASDs) comprise a range of disorders that share a core of neurobehavioural deficits characterized by widespread abnormalities in social interactions, deficits in communication as well as restricted interests and repetitive behaviours. The neurological basis and circuitry mechanisms underlying these abnormal behaviours are poorly understood. SHANK3 is a postsynaptic protein, whose disruption at the genetic level is thought to be responsible for the development of 22q13 deletion syndrome (Phelan-McDermid syndrome) and other non-syndromic ASDs. Here we show that mice with Shank3 gene deletions exhibit self-injurious repetitive grooming and deficits in social interaction. Cellular, electrophysiological and biochemical analyses uncovered defects at striatal synapses and cortico-striatal circuits in Shank3 mutant mice. Our findings demonstrate a critical role for SHANK3 in the normal development of neuronal connectivity and establish causality between a disruption in the Shank3 gene and the genesis of autistic-like behaviours in mice.     

肢带型肌营养不良一家系致病基因排除性定位

为了定位一个常染色体显性遗传肢带型肌营养不良家系的致病基因（ADLGMD）,采用13个荧光微卫星标记对收集到的一个包括4代33人的ADLGMD家系进行连锁分析,所选择的标记覆盖了3个已知ADL—GMD致病基因位点和4个已报道的致病基因定位区段．通过Linkage 5．1软件包计算连锁概率,各位点连锁分析所得的LOD值均小于-3,显示该家系致病基因与这7个位点均不连锁．该家系的肌营养不良症致病基因不在已知的位点内,很可能是一个新致病基因．     

The DNA sequence and analysis of human chromosome 13

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.     

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.     

A three-base-pair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa.

The group of retinopathies termed retinitis pigmentosa (RP) greatly contribute to visual dysfunction in man with a frequency of roughly 1 in 4,000. We mapped the first autosomal dominant RP (adRP) gene to chromosome 3q, close to the gene encoding rhodopsin, a rod photoreceptor pigment protein. Subsequently, mutations in this gene have been implicated as responsible for some forms of adRP. Another adRP gene has been mapped to chromosome 8p. A third adRP gene in a large Irish pedigree has been mapped to chromosome 6p, showing tight linkage with the gene for peripherin, a photoreceptor cell-specific glycoprotein, which is thus a strong candidate for the defective gene. We have now identified a three-base-pair deletion which results in the loss of one of a pair of highly conserved cysteine residues in the predicted third transmembrane domain of peripherin. This deletion segregates with the disease phenotype but is not present in unaffected controls, and suggests that mutant peripherin gives rise to retinitis pigmentosa.     

Mapping the limits of the human pseudoautosomal region and a candidate sequence for the male-determining gene

The human Y chromosome is composed of two different parts: a pseudoautosomal region shared with the X chromosome which is responsible for sex chromosome pairing and a Y-specific part that encodes the sex determining gene. Previously we have shown that the pseudoautosomal gene MIC2 only rarely recombines between the sex chromosomes and, based on the elevated recombination rates in the pseudoautosomal region, we predicted that this gene would lie close to the Y-specific region. In this report we describe a test of this prediction using long-range restriction mapping techniques. We conclude that MIC2 is less than 200 kilobases (kb) away from Y-specific sequences. During these experiments we have identified an HTF island in a position consistent with the proposed location of the human sex determining gene.     

Extra-embryonic function of Rb is essential for embryonic development and viability

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.     

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.