The Urokinase Receptor Is a Major Vitronectin-Binding Protein on Endothelial Cells

We have previously demonstrated that vitronectin (VN), a morphoregulatory protein in the vessel wall, is internalized and translocated to the subendothelial matrix by an integrin-independent mechanism (J. Histochem. Cytochem.41, 1823–1832, 1993). The cell surface component which mediates the initial contact of VN with endothelial cells is defined here. The specific binding of VN to endothelial cells demonstrated the following properties: a threefold increase after phorbol ester treatment; 85% inhibition by pretreatment of cells with phosphatidylinositol–phospholipase C to release glycolipid-anchored surface proteins; a 90% inhibition by urokinase (u-PA) receptor blocking antibody. u-PA increased VN binding to cells due to an eightfold increase in the affinity of VN for the u-PA receptor. Structure–function studies showed that the amino-terminal fragment of u-PA, devoid of any proteolytic activity, mediated this effect. Active plasminogen activator inhibitor-1 (PAI-1), but not inactivated PAI-1, inhibited VN binding to cells and displaced VN that was prebound to endothelial cell monolayers. Similarly, VN binding to purified (immobilized) u-PA receptor, but not to integrin, was enhanced by u-PA and inhibited by PAI-1. Hence, the binding of soluble VN to endothelial cell surfaces is mediated by the u-PA receptor, and the relative concentrations of u-PA and PAI-1 are able to regulate the strength of this interaction. Endothelial cell adhesion to immobilized VN was found to be integrin-mediated without any involvement of the VN–uPA-receptor system. Hence, the interaction of VN with the u-PA receptor may be involved in the regulation of cellular processes necessary for endothelial cell invasion and migration at VN-rich extracellular matrix sites.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995     

Cartographic study: Breakpoints in 1574 families carrying human reciprocal translocations

Reciprocal translocations (rcp) are among the most common constitutional chromosomal aberrations in man. Using a European database of 1574 families carrying autosomal rcp, a cartographic study was done on the breakpoints involved. The breakpoints are non-randomly distributed along the different chromosomes, indicating “hot spots”. Breakpoints of rcp that result in descendants that are unbalanced chromosomally at birth are more frequent in a distal position on chromosomal arms, and 65% of them are localised in R-bands. Among the R-bands, bands rich in GC islands and poor in Alu repetitive sequences are more frequently the site of breakpoints, as well as bands that include a fragile site. This result suggests that the variation in degree of methylation in GC islands could be involved in chromosomal breakage and hence in chromosomal rearrangements. Received: 10 April 1995 / Revised: 1 July 1995     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Assignment of the CD45-AP Gene to the Centromeric End of Mouse Chromosome 19 and Human Chromosome 11q13.1–q13.3

CD45-AP is a recently identified phosphorylated protein that specifically associates with the leukocyte-specific transmembrane glycoprotein CD45. The gene for CD45-AP,Ptprcap(protein tyrosine phosphatase, receptor type c polypeptide associated protein), was mapped in mouse by typing the progeny of two multilocus crosses using the mouse CD45-AP cDNA as a Southern hybridization probe. The CD45-AP gene mapped to the centromeric region of Chr 19 proximal to the genesFth, Cd5,andPcna-rs.The gene for the human CD45-AP homologue,PTPRCAP,was localized to chromosome band 11q13.1–q13.3 by fluorescencein situhybridization using human genomic CD45-AP DNA as a hybridization probe. The genetic mapping of thePtprcap/PTPRCAPgenes extends the previously defined synteny conservation of various genes that have been assigned to these regions of the mouse and the human chromosomes.     

Kanamycin rescue: A simple technique for the recovery of T-DNA flanking sequences

We have designed a new method for the recovery of T-DNA flanking sequences from T-DNA-tagged lines ofArabidopsis thaliana. Since most transformation vectors in use contain a plant-selectable marker for kanamycin resistance, we can use the 3′ part of thenptII coding region from the T-DNA to complement the bacterial 5′ region of thenptII gene from Tn5 to reconstruct a functional kanamycin-resistance gene inEscherichia coli. We have constructed a vector that contains the 5′ part of thenptII gene from Tn5 up to the uniquePst I site. By cloning total DNA from transformed lines in this vector, we were able to select directly for clones containing a T-DNA fragment, which reconstitutes a functional kanamycin gene, and a fragment of arabidopsis genomic DNA adjacent to the insertion. Flanking sequences up to 4 kb were rescued by this system.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.