Meltrin gamma(ADAM-9) mediates cellular adhesion through alpha(6)beta(1 )integrin, leading to a marked induction of fibroblast cell motility.

The ADAMs (A Disintegrin and Metalloprotease Domains) are a family of membrane-anchored proteins that play a role in fertilisation, myoblast fusion and ectodomain shedding of cell surface proteins. Meltrin gamma (ADAM-9) is a widely expressed member of this family and is involved in the shedding of heparin binding epidermal growth factor. Here we report that meltrin gamma can function as a cell adhesion molecule via its disintegrin domain. Using solid-phase binding assays and antibody inhibition experiments, we demonstrate that a murine meltrin gamma-Fc (Mel gamma -Fc) fusion protein binds to the integrin alpha(6)beta(1) on the surface of fibroblast cell lines, HT1080 and Wehi 164 in a specific manner. Since alpha(6)beta(1) is important for the motility of several cell types on laminin, cell migration studies using time-lapse video microscopy were performed. Cells adhering to Mel gamma-Fc displayed a rounded morphology and a marked increase (eight- to tenfold) in their motility compared to that on laminin. Furthermore, the p160 ROCK kinase inhibitor Y-27632 specifically reduced the migration of cells on meltrin gamma but had no effect on migration of cells on laminin, whilst the general tyrosine phoshorylation inhibitor, genistein, inhibited cell migration on both substrates. These results together suggest that meltrin gamma may play a role in regulating the motility of cells by binding to alpha(6)beta(1) integrin and this may be important during a variety of biological and pathological processes.

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.

Interaction of metargidin (ADAM-15) with alphavbeta3 and alpha5beta1 integrins on different haemopoietic cells.

Metargidin (ADAM-15) is a type I transmembrane glycoprotein belonging to the ADAM (A Disintegrin and Metalloprotease Domain) family of proteins and is widely expressed in different tissues and cell types. Members of this family contain an amino-terminal metalloprotease domain followed by a disintegrin domain, a cysteine-rich region and a membrane proximal EGF-like domain. The disintegrin domain of metargidin contains an RGD tripeptide sequence, suggesting that it may potentially interact with the integrin family of proteins. Here we identify integrin ligands for metargidin on haemopoietic cells, by using a chimeric protein containing the extracellular domain of metargidin fused to the Fc portion of human IgG. Binding activity to a panel of human cell lines was analysed by solid-phase cell-adhesion assays. Metargidin bound to a monocytic cell line, U937, and a T cell line, MOLT-4, in a specific manner. Adhesion was divalent cation- and temperature- dependent and strongly enhanced by Mn2+, all features of integrin-mediated binding. Using a panel of anti-integrin antibodies we show that alphavbeta3 is a ligand for metargidin on U937 cells. In contrast, for MOLT-4 cells, the integrin alpha5beta1 contributes to cell binding. Adhesion was mediated by the disintegrin domain of metargidin as RGD-based peptides inhibited cell binding to both cell lines. The specificity of the interaction between both alphavbeta3 and alpha5beta1 and metargidin was further confirmed by solid-phase adhesion assays using purified recombinant integrins. These results together indicate that metargidin can function as a cell adhesion molecule via interactions with alphavbeta3 and alpha5beta1 integrins.

TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3.

TNF-alpha converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-alpha to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-alpha secretion is involved.

The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes.

We report that monolayers of human fibroblasts stimulated with concanavalin A were able to activate 72 kDa progelatinase but not 95 kDa progelatinase. The activating capacity of fibroblasts appeared approx. 6 h after concanavalin A stimulation and was blocked by cycloheximide. The activation of 72 kDa progelatinase was readily inhibited by TIMP-2 but only poorly by TIMP-1. Plasma membranes isolated from the fibroblasts were capable of activating 72 kDa progelatinase. The cleavage products of the plasma membrane-mediated activation of 72 kDa progelatinase corresponded to those of organomercurial-induced self-cleavage. Only inhibitors of metalloproteinase self-cleavage inhibited the activating capacity of plasma membrane preparations, although the activating capacity was destroyed by trypsin and heat. As with the fibroblast monolayers, TIMP-2 was a potent inhibitor of the membrane-mediated activation whereas TIMP-1 was less so.

Differences in the expression of the human interferon-gamma gene in fresh lymphocytes and cultured lymphoblasts.

Fresh human peripheral blood mononuclear lymphocytes and lymphoblasts that had been grown for a period in T-cell growth-factor containing medium were stimulated with staphylococcal enterotoxin A plus mezerein to produce interferon-gamma (IFN-gamma). Growing lymphoblasts produced peak levels of IFN-gamma much earlier after induction than fresh lymphocytes. Quantitation of the steady-state levels of IFN-gamma mRNA showed these to differ markedly between the two cell types over a period of time post-induction. In fresh lymphocytes the steady-state levels of IFN-gamma mRNA increased to a peak level over a period of 4 days while in growing lymphoblasts the peak level occurred after 8 hours. These differences in IFN-gamma mRNA production were shown to be not the result of gross alteration of RNA metabolism following blast transformation.

A comparison of vertebrate interferon gene families detected by hybridization with human interferon DNA.

Cloned human interferon complementary DNAs were used as hybridization probes to detect interferon alpha and beta gene families in restriction endonuclease digests of total genomic DNA isolated from a wide range of vertebrates and invertebrates. A complex interferon-alpha multigene family was detected in all mammals examined, whereas there was little or no cross-hybridization of human interferon-alpha complementary DNA to non-mammalian vertebrates or invertebrates. In contrast, human interferon-beta complementary DNA detected one or two interferon-beta genes in all mammals tested, with the exception of the cow and the blackbuck, both of which possessed a complex interferon-beta multigene family which has presumably arisen by a recent series of gene duplications. Interferon-beta sequences could also be detected in non-mammalian vertebrates ranging from birds to bony fish. Detailed restriction endonuclease mapping of DNA sequences neighbouring the interferon-beta gene in a variety of primates indicated a strong evolutionary conservation of flanking sequences, particularly on the 3' side of the gene.

Plasmid replication functions. IV. Promoters in the replication region of plasmid R6-5.

Eight RNA polymerase binding sites have been shown to map within the EcoRI fragment E-2 (replication region RepA EcoRI fragment) of plasmid R6-5 and all but one have been shown to contain active promoters of transcription. Three of the identified promoters are located within a 2.7 kb region essential for controlled, autonomous plasmid replication and may be involved in the functional expression of the three R6-5 replication determinants that have thus far been identified, namely the origin of vegetative replication, oriV, the replication control gene, cop, and the determinant of an essential, positive-acting element, designated RepA.

Plasmid replication functions. II. Cloning analysis of the repA replication region of antibiotic resistance plasmid R6-5.

R6-5 is a low copy number, conjugative, FII incompatibility group plasmid that has a molecular length of 102 kb and that specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts. By means of in vitro cloning procedures, mini plasmids have been generated that contain a DNA segment from the essential region of R6-5 that is only 2.6 kb in length. This DNA segment, which consists of two PstI fragments that are adjacent in the parent plasmid, carries all genes and sequences required for the regulated replication and incompatibility properties of R6-5, including its origin of replication, OriV, an essential function that has been designated RepA, and the copy control function, Cop. Three different polypeptides, having monomer molecular weights of 23,000, 10,000 and 9,500 daltons, are synthesized in detectable quantities by minicells carrying pBR322 hybrid plasmids that contain DNA segments from the R6-5 essential region. A spontaneous deletion derivative of a pBR322 hybrid plasmid that carries the R6-5 origin of replication was isolated. Heteroduplex analysis of this derivative plasmid indicates that the deleted DNA segment carries the R6-5 replication origin and that its termini consist of short inverted repeat sequences.

Plasmid incompatibility: cloning analysis of an incFII determinant of R6-5.

SalI and PstI restriction endonuclease-generated DNA fragments that specify an FII-type incompatibility function (incFII) of the low copy number antibiotic resistance plasmid R6-5 have been cloned in the high copy number pBR322 plasmid vector. A 1-kilobase DNA sequence that contains this incFII determinant has been identified and is shown to have coordinates of 95.5 and 96.5 kilobases on the R6-5 plasmid physical map. Expression of incompatibility by the cloned PstI fragment depends on its orientation within the vector molecule. The behaviour of pBR322-incFII hybrid plasmids suggests that plasmid replication control is not the only mechanism that can cause incompatibility between two plasmids.

Nucleotide sequence of bacteriophage phi X174 DNA.

A DNA sequence for the genome of bacteriophage phi X174 of approximately 5,375 nucleotides has been determined using the rapid and simple 'plus and minus' method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.

A restriction cleavage map of phiX 174 DNA by pulse-chase labelling using E. coli DNA polymerase.

A pulse chase labelling technique using E. coli DNA polymerase I has been used to determine a number of restriction endonuclease cleavage maps of ØX 174 DNA. In addition to verifying the accuracy of the method by confirming some previously published maps, the Hha I and Alu I maps have also been constructed.Images