Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena.

The codon usage of Tetrahymena thermophila and other ciliates deviates from the 'universal genetic code' in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.     

A Bioluminescence Method for the Measurement of l-Glutamate: Applications to the Study of Changes in the Release of l-Glutamate from Lateral Geniculate Nucleus and Superior Colliculus After Visual Cortex Ablation in Rats

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.     

An intron in a ribosomal protein gene from Tetrahymena

We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact that it is found together with the well described self-splicing rRNA intron is discussed in relation to the evolution of RNA splicing.     

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification

Hybridomas producing a monoclonal IgG1 antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66,000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified approximately 200-fold from conditioned medium from the melanoma cells with a yield of 79%. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 125I-labelled HPA66 was approximately 2.5 x 10(9) l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.     

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.     

Effects on microbial activity by extraction of indigenous cells from soil slurries

Abstract: Possible effects on the physiological activity and culturability of soil microorganisms by different soil dispersion procedures, and effects on activity caused by extracting bacteria from soil, were investigated. There was no apparent difference in cfu's with dispersion of a silty loam soil and a loamy sand soil with pyrophosphate as compared to dispersion in NaCl. Substrate-induced respiration was reduced in the silty loam soil, and methanol oxidation was reduced in the loamy sand soil with dispersion in pyrophosphate, and the soil pH was irreversibly increased by the treatment. Extracted bacterial fractions had lower numbers of culturable cells as percentage of the total number of bacteria in each fraction, lower respiration rates and no methanol oxidation activity as compared to the soil slurry both before and after extraction. The physiological activity was apparently not affected by the number of cells extracted. This indicates that the increased extraction rate of indigenous soil bacteria obtained by effective disruption of aggregates and detachment of cells from surfaces, only results in increased extraction of cells that have been physiologically changed as a result of the extraction process.     

Phosphorylation of glycogen synthase in a homogenate of human polymorphonuclear leukocytes

Summary Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the³²P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the³²P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). One P_i/subunit was incorporated within 8–10 min and 2.2 P_i/subunit within 60 min increasing the K_c for Gle-6-P to 4–6 mM. The initial phosphorylation up to 0.8 P_i/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the interval RI 0.85 to RI 0.05 and phosphorylation of this peptide to 0.5 P_i was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence. From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from phosphorylase kinase is presented. The endogenous Gle-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 P_i/subunit and K_c for Glc-6-P was increased from 6–8 mM to 20 mM. From the present and previous experiments [7] a total of 8 major phosphorylatable sites have been defined, one on each of the peptides t-A, t-B, t-C, c-B and c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The operational defined synthase R (K_c for Glc-6-P 0.5 mM) and D (K_c for Glc-6-P 2–8 mM) activities correspond to synthase with about 0.8 P_i and 1.8–2.3 P_i/subunit, respectively.     

Receptors for the complement C3d component and the Epstein-Barr virus are quantitatively coexpressed on a series of B-cell lines and their derived somatic cell hybrids

Various human Burkitt lymphoma and LCL lines established in vitro and their derived somatic cell hybrids were tested for their comparative EBV receptor levels in a virus binding assay. Their graded C3b and C3d complement receptor expression was estimated simultaneously by means of isotope labeled rosette marker cells. The receptor concentration of each cell line was related to Raji as the standard of comparison, K 562, P3HR-1, and YACUT were used as negative controls. In general, the charging curves for EBV and C3d receptors parallelled each other (r = 0.97) while C3b receptor charging showed no correlation (r < 0.60). In the Raji hybrids between the C3b receptor positive Raji parent and various patents that were negative for this receptor, C3b receptor expression was low or negative. In contrast, the C3d negative P3HR-1 line gave rise to hybrids, after fusion with receptor-positive cells, that were intermediate with regard to their C3d receptor expression. The host range restriction of the Epstein-Barr virus is determined at the receptor level. The close relationship between the EBV receptor and the C3d receptor, a B-lymphocyte-specific moiety, suggests that the moderate interaction with EBV with the B lymphocytes may have had a selective advantage, favoring the presence of EBV. Since EBV causes lytic infections after artificial introduction into nonnatural host cells, it may represent a B-lymphocyte-specific host range mutant, derived from an originally lytic herpesvirus with a much broader target cell range.     

Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies.

Type-1 inhibitor of plasminogen activators (PAI-1) occurs in purified preparations in a latent form that can be activated with denaturants; in vivo, latency is prevented by binding to vitronectin. We have compared latent, denaturant-activated and reactive centre-cleaved human PAI-1 with respect to thermal stability and affinity to monoclonal antibodies. By both criteria, latent and cleaved PAI-1 are very similar or indistinguishable, and clearly different from active PAI-1. Our findings suggest that the conformations of latent and reactive centre-cleaved PAI-1 are similar and resemble the so-called relaxed (R) serpin conformation, while that of active PAI-1 is different and resembles the stressed (S) serpin conformation.