Slits and Roundabouts in cancer, tumour angiogenesis and endothelial cell migration.

Angiogenesis describes the development of new blood vessels from pre-existing vessels. The hijacking of this physiological process by tumours allows them to develop their own supplies of nutrients and oxygen, enabling their growth and metastasis. A large body of literature has accumulated over the last 20 years relating to angiogenesis, including signalling pathways involved in this process.

The thioredoxin-like fold: hidden domains in protein disulfide isomerases and other chaperone proteins.

Although protein disulphide isomerase (PDI) has been known for nearly 40 years, several new PDIs have recently been described that reveal a remarkable diversity in both structure and function. This article reviews our current knowledge of the PDI family members and identifies four novel PDIs in the human genome. These include human transmembrane proteins that have C.

JmjC: cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta.

On the basis of significant sequence similarity, we have identified JmjC domains in more than 100 eukaryotic and bacterial sequences. These include human hairless, mutated in individuals with alopecia universalis, retinoblastoma-binding protein 2 and several putative chromatin-associated proteins. JmjC domains are predicted to be metalloenzymes that adopt the cupin fold, and are candidates for enzymes that regulate chromatin remodelling.

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease.

Expression of beta-catenin and the adenomatous polyposis coli tumour suppressor protein in mouse neocortical cells in vitro.

Beta-catenin is known to associate with the tumour suppressor protein adenomatous polyposis coli (APC), which is highly expressed in developing brain. We have therefore investigated the distribution of beta-catenin and APC in primary cultures of mouse neocortex. Western blotting demonstrated the presence of a single beta-catenin species in our cultures.

The cellular distribution of the adenomatous polyposis coli tumour suppressor protein in neuroblastoma cells is regulated by microtubule dynamics.

The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein.

Yeast artificial chromosome cloning of the beta-catenin locus on human chromosome 3p21-22.

beta-Catenin has emerged as an important component of the adherens junctions between epithelial cells. As a result of studies of its interaction with the APC gene product, it has been implicated in the development of colorectal cancer. alpha-Catenin, beta-catenin, E-cadherin and APC appear to mediate contact inhibition in epithelia.

Simultaneous detection of amplicon and HSV-1 helper encoded proteins reveals that neurons and astrocytoma cells do express amplicon-borne transgenes in the absence of synthesis of virus immediate early proteins.

HSV-1 amplicon vectors were used to express either a cytoplasmic (beta-galactosidase) or a membrane targeted protein (TIMP-Thy1) in primary neuronal cultures, and a human astrocytoma cell line. Whereas some cells became infected by vector particles alone others were simultaneously infected by both vector and helper particles. Our results show that IEHCMV and HSV-1 IE3 promoters are able to direct transgene expression in these cells in the absence of synthesis of helper virus transacting proteins, and stress the need of monitoring expression from both partners of an amplicon population, in order to differentiate transgene expression in cells singly infected with amplicon particles, from those infected by both amplicon and helper particles.

Recombinant glycosyl-phosphatidylinositol-anchored proteins are not associated with protein kinases in transfected thymoma cells.

The cross-linking by antibody of some glycosyl-phosphatidyl-inositol (GPI)-anchored proteins on the plasma membrane of T cells leads to cell activation. Phosphorylation of proteins on tyrosine residues has a central role in the control of T cell activation, and non-receptor protein tyrosine kinases can be coprecipitated with immune complexes of GPI-anchored proteins in T cell lysates. In order to investigate the nature of this interaction, two recombinant GPI-anchored proteins were constructed (using the GPI signal sequence from Thy-1), and their associations with protein tyrosine kinases in stable transfectants of a mouse thymoma have been investigated.

Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia.

HSV-1 derived amplicons expressing cytoplasmic beta-galactosidase (pA-SF1), or plasma membrane targeted TIMP-Thy1 (pA-TT1), were used to transduce glial cells in vitro. By monitoring the expression of reporter genes from both amplicons and helper virus, we determined that many cells were infected by both particles. In glial cells infected only by pA-SF1 beta-galactosidase immunoreactivity was restricted to the cytoplasm; co-infection with helper HSV-1 (wild type), resulted in additional nuclear beta-galactosidase immunoreactivity.

Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-term expression of transgenes encoding cytoplasmic and membrane anchored proteins in postmitotic polarized cortical neurons and glial cells in vitro.

We constructed three recombinant vectors derived from the herpes simplex virus type 1 mutant tsK, each of which contained a different transgene under the control of the herpes simplex virus type 1 immediate early 3 promoter inserted into the thymidine kinase locus: the prokaryotic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloproteinases linked to the last exon of Thy-1, which encodes for a glycosyl-phosphatidyl-inositol membrane anchor. Infection of postmitotic neocortical and hippocampal neurons in low-density primary cultures with these vectors, achieved reliable expression of all three foreign gene products in various neocortical cell types, e.g.

HSV1 vectors to study protein targeting in neurones: are glycosyl-phosphatidylinositol anchors polarized targeting signals in neurones?

In order to characterize protein targeting signals in polarized postmitotic cortical neurones in vitro, we have developed recombinant and amplicon type vectors derived from herpes simplex virus 1 (HSV1) to transfer genes into these cells. We examined the targeting of both bacterial proteins, which lack specific targeting signals, as well as recombinant proteins containing mammalian targeting sequences, i.e.

Construction, expression and functional analysis of a glycolipid-linked form of CR1.

By genetic engineering of human CR1 cDNA and its stable transfection into cells we have produced a cell line which expresses CR1 anchored to the cell surface by a glycolipid anchor. The glycosyl-phosphatidylinositol (GPI)-CR1 protects cells intrinsically from damage mediated by complement activated through the classical pathway. Cell surface GPI-CR1 is more efficient on a molar basis than soluble CR1 in the assay, but extrinsic protection of other cells was not obtained.

A cDNA construct of tissue inhibitor of metalloproteinases (TIMP) linked to the last exon of Thy-1 confers glycophospholipid anchorage on this naturally secreted protein.

A naturally secreted protein, tissue inhibitor of metalloproteinases (TIMP), has been transiently expressed on the surface of transfected COS cells and stably on transfected murine BW 5147 thymoma cells, by linkage of the entire coding sequence of the cDNA to the last exon of Thy-1. Thy-1 is a glycophospholipid-linked protein. In COS cells the chimaeric protein can be labelled by [3H]ethanolamine, which is a component of glycophospholipid anchors.

A novel strategy for producing chimeric bispecific antibodies by gene transfection.

We have used genetic manipulation to produce chimeric bispecific antibodies. Plasmids containing variable regions of immunoglobulin from a murine hybridoma secreting anti-hepatitis B surface antigen were joined to human constant regions. These chimeric plasmids were introduced into transfectomas, secreting chimeric antibodies to iodo-hydroxy-nitrophenyl, by electroporation.

The difference between human C3F and C3S results from a single amino acid change from an asparagine to an aspartate residue at position 1216 on the alpha-chain of the complement component, C3.

The third component of the human C system, C3, exhibits two common genetic variants. These variants have been characterized by high voltage agarose electrophoresis and are designated C3 fast (C3F) and C3 slow (C3S). C3F occurs at appreciable frequencies only in Caucasian populations and has been shown to be associated with an increased incidence of certain diseases, such as partial lipodystrophy, IgA nephropathy, and Indian childhood cirrhosis.

Chemical synthesis, cloning and expression in mammalian cells of a gene coding for human tissue-type plasminogen activator.

A 1610-bp DNA duplex coding for human tissue-type plasminogen activator has been chemically synthesized using the phosphoramidite procedure, adapted for a custom-built gene synthesizer. The synthesizer, which was designed for both simplicity and speed, permits the rapid construction of relatively large genes and compares favorably in speed with alternative cDNA isolation procedures. The plasminogen activator gene has been expressed in mammalian cells and shown to produce authentic protein by an immuno-activity assay.

Structure of mouse major urinary protein genes: different splicing configurations in the 3'-non-coding region.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3'-non-coding region and contain different polyadenylation sites.

Messenger RNAs coding for mouse major urinary proteins are differentially induced by testosterone.

We have investigated the sexual dimorphism of the mouse major urinary proteins (MUPs) by isoelectric focusing (IEF). In each of two inbred strains which have different male patterns (C57BL and BALB/c), the females show a simpler pattern with fewer prominent components. The main female component is different in each strain, and these may be the products of allelic structural genes.

Variation between mouse major urinary protein genes isolated from a single inbred line.

We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines.