First clinical evaluation of a new concept for puncture-site occlusion in interventional cardiology and angioplasty.

UNLABELLED: Percutaneous transluminal angioplasties of coronary and peripheral vessels are frequently used and replace open surgery in a certain percentage. Hemostasis in most of these patients is reduced or inhibited and often leads to hemorrhage from the puncture track. Due to this fact, hospitalization is not only mandatory, but also surgical revision of the puncture site is often required. We designed and produced a coaxial delivery system, which is mounted on the indwelling guide-wire after withdrawal of the instrumentation for angioplasty. The delivery system is advanced down to the outer wall of the punctured vessel and 1 cc of human two-compound fibrin glue is released. Based on our experience with laboratory and animal research, which we already presented at the 7th International Symposium on Pediatric Surgical Research in Heidelberg, May 27-28, 1994, we conducted the first trials in interventional cardiology. In 1996, a first group of 10 patients, aged 49 to 80 years, underwent sealing of the right femoral artery after diagnostic evaluation (n = 3) of coronary balloon dilatation (n = 7). In patients, the local manual compression time was less than 5 minutes and 1 patient needed 10 minutes of digital compression. In one case, bleeding continued and a compression bandage was successful, whereas in another case the local hematoma formation needed surgical revision with suture of the ruptured vessel wall. CONCLUSION: Puncture-track sealing with locally applied fibrin glue seems to be an efficient tool to avoid bleeding after interventions of coronary and peripheral vessels. In the meantime, the device has been improved by a target system to optimize the delivery of the glue exactly at the outer wall of the vessel.

The metastasis suppressor candidate nucleotide diphosphate kinase NM23 specifically interacts with members of the ROR/RZR nuclear orphan receptor subfamily.

We have cloned proteins that interact with the nuclear orphan receptor RZR beta using the yeast two-hybrid system. We identified, amongst a number of other genes, the nucleoside diphosphate kinase (NDPK)-2 also known as Nm23-2, c-myc regulatory factor PuF and differentiation inhibitory factor, RZR beta specifically interacts with Nm23-2 but not with the closely related tumor metastasis suppressor candidate gene product Nm23-1. In contrast ROR alpha interacts with both Nm23 proteins. These findings were corroborated by in vitro interaction assays based on GST-pulldown experiments. With-n-myc we propose a candidate gene regulated by ROR alpha/RZR beta and Nm23, based on the finding that the respective DNA binding sites in the first intron are conserved in several mammalian species.

Mechanism of irreversible inactivation of phosphomannose isomerases by silver ions and flamazine.

Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have previously shown that the enzyme phosphomannose isomerase (PMI) is essential for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is shown to be irreversible, and is a two-step process, involving an intermediate complex with a dissociation constant, Ki, of 59 +/- 8 microM, and a maximum rate of inactivation of 0.25 +/- 0.04 min-1 in 50 mM Hepes buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissociation constant of 0.31 +/- 0.04 mM, close to its Km value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compared the ability of silver sulfadiazine and two other silver-containing compounds to irreversibly inactivate C. albicans PMI. The addition of the organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a Ki of 190 +/- 30 nM. In all cases, the maximum inhibition rate was similar, implying a similar rate-determining step. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivation process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed similar Vm and Km values to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product.

The Saccharomyces cerevisiae PKC1 gene encodes a homolog of mammalian protein kinase C (Levin, D. E., Fields, F.O., Kunisawa, R., Bishop, J.M., and Thorner, J. (1990) Cell 62, 213-224). A protein of 150 kDa is recognized by a polyclonal antiserum raised against a trpE-Pkc1 fusion protein. In subcellular fractionations, Pkc1p associates with the 100,000 x g particulate fraction. This association is resistant to extraction with high salt concentrations, alkali buffer, or nonionic detergents, suggesting that Pkc1p may be associated with a large protein complex. Pkc1p modified at its COOH terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ sequence tag) was able to fully restore the growth defects of a pkc1ts strain at restrictive temperature. ZZ-tagged Pkc1p was partially purified by chromatography on DEAE-Sepharose, followed by IgG-Sepharose. In vitro, Pkc1p phosphorylates the pseudosubstrate peptide and myelin basic protein, but not histones. Replacing an isoleucine with an arginine 2 amino acids COOH-terminal of the acceptor serine in the substrate peptide resulted in a 10-fold decrease of Km. Pkc1p activity was independent of cofactors such as phospholipids, diacylglycerol, and Ca2+, known to activate several mammalian protein kinase C isoenzymes, making it a rather distantly related member of the protein kinase C superfamily.

The TRP4 gene of Saccharomyces cerevisiae: isolation and structural analysis.

The TRP4 gene of Saccharomyces cerevisiae, encoding the anthranilate phosphoribosyl transferase, was isolated and subcloned by functional complementation in yeast. A 2 kb fragment containing information for a polypeptide of 380 amino acids and the 5'- and 3'-flanking regions was sequenced. The TRP4 transcript was identified and mapped with S1 nuclease. Homologies to two prokaryotic genes encoding the same function, and sequences potentially involved in transcription start and termination and in regulation of TRP4 gene expression are discussed.