Cyclic 3',5'-adenosine monophosphate in the choroid plexus: stimulation by cholera toxin.

Cholera toxin was found to induce high accumulations of cyclic AMP in the isolated choroid plexus of the rabbit and in the incubation medium. The accumulation showed a characteristic lag phase of at least 30 min and continued for at least 3 hours. Inactivated cholera toxin was unable to increase cyclic AMP levels. There was only a moderate effect of cholera toxin on cyclic AMP “low K_m” phosphodiesterase activity in homogenates. The effect of cholera toxin on cyclic AMP levels confirms the existance of a potent cyclic AMP generating system in the choroid plexus which is activated also by β-adrenergic agonists, histamine and prostaglandin E₁.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture

Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen produced is cartilage specific collagen (type II). This work was supported in part by grant HD-05505 from NIH.     

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).     

Deuterium order parameters in relation to thermodynamic properties of a phospholiped bilayer. A statistical mechanical interpretation.

The physical properties of bilayers of dipalmitoyl-3-sn-phosphatidylcholine are analyzed in terms of a statistical model proposed by Marcelja (S. Marcelja (1974), Biochim. Biophys. Acta 367, 165). The model is used to calculate the segmental order parameters of the hydrocarbon chains, the transition temperature of the crystalline leads to liquid crystalline phase transition, the entropy change of the transition, the bilayer thickness, and the linear thermal expansion coefficient. The theoretical predictions are in excellent agreement with experimental results obtained by deuterium magnetic resonance, differential scanning calorimetry, and X-ray diffraction. The model yields the probabilities of trans and gauche conformations and also those of more specific conformational defects like kinks or jogs.     

Differential fluorescence labelling with 5-dimethyl-aminonaphthalene-1-sulfonyl chloride of intact cells and isolated membranes in Salmonella typhimurium and Acholeplasma laidlawii

For fluorescence labelling intact cells and isolated cell envelopes (membranes) from Salmonella typhimurium and Acholeplasma laidlawii were treated with mixed dansylchloride-lecithin-cholesterol vesicles. This kind of dansylation, which has been supposed to be specific for cell surface proteins, produced fluorescent protein pattern after SDS-polyacrylamide gel electrophoresis only when isolated envelopes were dansylated. Acid hydrolysis of fluorescent cell envelopes of Salmonella typhimurium yielded O-dansyltryosine and epsilon-N-dansyl-lysine besides the free sulfonic acid and unidentified compounds. However, no fluorescent proteins were detectable in cell envelopes isolated from dansylated intact bacteria from Salmonella typhimurium. In accord Acholeplasma laidlawii showed only fluorescence from proteins with a molecular weight higher than 100000.     

A modified dinucleotide motif specifies tRNA recognition by TLR7

RNA can function as a pathogen-associated molecular pattern (PAMP) whose recognition by the innate immune system alerts the body to an impending microbial infection. The recognition of tRNA as either self or nonself RNA by TLR7 depends on its modification patterns. In particular, it is known that the presence of a ribose methylated guanosine at position 18, which is overrepresented in self-RNA, antagonizes an immune response. Here, we report that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The most efficient nucleobases combination of this motif includes two purines, while pyrimidines diminish the effect of ribose methylation. The constraints of this motif stay intact when transposed to other parts of the tRNA. The results argue against a fixed orientation of the tRNA during interaction with TLR7 and, rather, suggest a processive type of inspection.