Amiloride selectively inhibits the urokinase-type plasminogen activator

The diuretic drug amiloride, an inhibitor of Na+ uptake, competitively inhibits the catalytic activity of the urokinase-type plasminogen activator (u-PA), with a Ki of 7 X 10(-6) M. Generation of plasmin, cleavage of peptide substrates, and interaction of u-PA with a specific macromolecular proteinase inhibitor are all prevented in the presence of the drug. In contrast, amiloride does not affect the activity of either tissue-type plasminogen activator, plasmin, plasma kallikrein or thrombin. The inhibition of u-PA by amiloride may be related to the previously reported inhibition of u-PA-type enzymes by Na+. Amiloride or related compounds could prove useful in selectively controlling u-PA-catalyzed extracellular proteolysis.     

A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase

The secretion of plasminogen activators has been implicated in the controlled extracellular proteolysis that accompanies cell migration and tissue remodeling. We found that the human plasminogen activator urokinase (Uk) (Mr 55,000 form) binds rapidly, specifically, and with high affinity to fresh human blood monocytes and to cells of the monocyte line U937. Upon binding Mr 55,000 Uk was observed to confer high plasminogen activator activity to the cells. Binding of the enzyme did not require a functional catalytic site (located on the B chain of the protein) but did require the noncatalytic A chain of Mr 55,000 Uk, since Mr 33,000 Uk did not bind. These results demonstrate the presence of a membrane receptor for Uk on monocytes and show a hitherto unknown function for the A chain of Uk: binding of secreted enzyme to its receptor results in Uk acting as a membrane protease. This localizes plasminogen activation near the cell surface, an optimal site to facilitate cell migration.     

A simple protocol for extraction and detection of pyrazines produced by fungi in solid state fermentation

Summary A protocol for the extraction and detection of pyrazines produced by fungi in Solid State Fermentation that gave good recovery of these compounds was devised together with specific techniques for detection by Gas Chromatography (GC). The protocol involved solid-liquid and liquid-liquid extractions, concentration of the extracts and GC analysis. Extraction yields were measured from a model system in which synthetic pyrazines were added. Extracts were analysed with a gas chromatograph equipped with a thermo-ionic detector to allow specific pyrazine detection.     

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

The signal sequence of the murine serine protease inhibitor PAI-2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild-type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI-2 signal sequence. Using a malE signal sequence mutant, which has a Mal-phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele-specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG.     

Pharmacological effects of GABA on serotonin metabolism in the rat brain

The pharmacological effects of GABA-related drugs were studied on the serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA) contents of various regions of the rat brain. These effects were examined in the nuclei raphe dorsalis, magnus and centralis and in structures receiving a dense serotonin innervation such as the habenula complex and subcommissural organ. The GABA agonist, muscimol, increased the 5-HT contents and reduced 5-HIAA levels in structures containing serotoninergic terminals suggesting an inhibitory effect of GABA on the firing of serotoninergic neurons with concomitant reduction of 5-HT utilisation. In contrast, the GABA antagonist, bicuculline, probably stimulated 5-HT turnover since its intraperitoneally administration produced significant increase of 5-HT and/or 5-HIAA levels in the same brain regions. These data are in agreement with a transsynaptic inhibitory control of GABA on serotoninergic neurons. Drugs which inhibit the GABA catabolism such as amino-oxyacetic acid or gamma-vinyl-GABA and which should elevate GABA levels in the synaptic gap were capable of increasing or decreasing the 5-HT and the 5-HIAA levels depending on the experimental conditions. These results suggest that several processes are probably involved in the control of serotoninergic neurons by GABA in the rat brain. Among them, an intracellular effect of GABA on 5-HT metabolism might well occur in cells containing both GABA and 5-HT.     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.     

A study of the budding ofSaccharomyces uvarum Beijerinck with the scanning electron microscope

Vegetative cells ofSaccharomyces uvarum Beijerinck in the exponential growth phase were examined with the scanning electron microscope. The existence of two types of scars — birth scars and bud scars — was confirmed. Birth scars had larger diameters than bud scars; both remained visible on old cells. The distribution of the buds on the mother cell did not appear to be a random one: there seemed to be a more or less emphasized cell polarity. The author wishes to thank Mr. Bert for technical assistance in the use of the scanning electron microscope.