The effect of low sulfate concentrations on the glycosaminoglycan synthesis in anatomically intact articular cartilage of the mouse

We have studied the effect of environmental sulfate concentration on the glycosaminoglycan synthesis of anatomically intact patellar cartilage of the mouse in vitro. Incubation of mouse patellae in medium with sulfate concentrations below 0.5 mM resulted in a diminished incorporation of sulfate but in unaltered incorporation of glucosamine. This suggested the synthesis of undersulfated glycosaminoglycans under these conditions. We characterized glycosaminoglycans synthesized at three different sulfate concentrations: a sulfate concentration physiological for the mouse (1.0 mM), a sulfate concentration in the range where sulfate incorporation was strongly diminished (0.1 mM), and an extremely low sulfate concentration (10 nM). Analysis of glycosaminoglycan disaccharides and DEAE anion chromatography of the glycosaminoglycans could not confirm the synthesis of undersulfated glycosaminoglycans at 0.1 mM. The chromatogram of glycosaminoglycans synthesized in medium containing 10 nM showed the presence of a very low sulfated glycosaminoglycan pool not observed at higher medium sulfate concentrations. Intermediately sulfated glycosaminoglycans were also synthesized during incubation with 10 nM sulfate. So, our data indicate that only very low sulfate concentrations in the medium lead to the synthesis of undersulfated glycosaminoglycans and that the sulfation mechanism of murine patellar cartilage chondrocytes does not seem to fit completely in an "all-or-nothing" pattern.     

Lipophilic analogues of sparsomycin as strong inhibitors of protein synthesis and tumor growth: a structure-activity relationship study

Fourteen derivatives of sparsomycin (1) were synthesized. Six of them were prepared following a novel synthetic route starting from the L-amino acid alanine. Some physicochemical properties, viz. lipophilicity and water solubility, of selected derivatives were measured. The biological activity was tested in vitro in cell-free protein synthesis inhibition assays, in bacterial and tumor cell growth inhibition assays, and in the L1210 leukemia in vivo model in mice. Also for selected drugs the acute toxicity in mice was determined. Ribosomes from both an eukaryotic and a prokaryotic organism were used in the protein synthesis inhibition systems. A linear correlation between the lipophilicity parameters measured was observed. Water solubility and drug toxicity in mice were found to be linearly correlated with lipophilicity. All the derivatives studied are more lipophilic than 1. The deshydroxysparsomycin analogues (30-33) showed an interesting phenomenon: increase in hydrophobicity was accompanied by a considerable increase in water solubility. We found that an increase in hydrophobicity of the drug as a result of replacing the SMe group of 1 with larger alkylthio groups causes an increase in the biological activity of the drug. However, not only the hydrophobicity but also shape and size of the substituent are important; in the homologous series 1-9-10-11-12, 21-22-23-24, and 30-31-32-33, highest protein synthesis inhibitory and in vitro cytostatic activity is found with compounds 11, 23, and 32, respectively, and in comparison with the highly active n-butyl compound 10, the isomeric tert-butyl compound 13 is rather inactive. Polar substituents replacing the SMe group, i.e. Cl in 17 and 35, also render the molecule inactive. Substituting the bivalent sulfur atom for a methylene group decreases the drug's activity. This effect can be compensated for by increasing the length of the alkylsulfinyl side chain. The agreement between the results derived from cell-free and "in vivo" tests is good. The assays using ribosomes of bacterial and eukaryotic organisms give similar results although the latter seem to be more sensitive to changes in hydrophobicity of the drug. Our results confirm the presence of a hydrophobic region at the peptidyl transferase center of the ribosome; the interaction of sparsomycin with this region is more pronounced in the eukaryotic particles. The sparsomycin analogues 11, 23, and 30 show the highest antitumor activity against L1210 leukemia in mice, their median T/C values are 386, 330, and 216%, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitochondrial Antibodies in Primary Biliary Cirrhosis: II. The Complement Fixing Antigen as a Component of Mitochondrial Inner Membranes

The autoantibodies found in the sera of patients with primary biliary cirrhosis have been shown to react with a component of the mitochondrial inner membranes. Outer membranes were inactive. The purity of the inner and outer membrane fractions obtained by 2 different methods was assessed by electron microscopy and marker enzyme tests. Using negative-staining it was possible to visualize antibody binding to mitochondrial membranes. At high resolution it could be seen that the 90° particles on the cristal membranes were not involved in the reaction with antibody, but it was not possible to establish in the present studies the precise antigenic site upon the mitochondrial inner membranes.     

Neuromuscular defects in a Drosophila survival motor neuron gene mutant

Autosomal recessive spinal muscular atrophy (SMA) is linked to mutations in the survival motor neuron (SMN) gene. The SMN protein has been implicated at several levels of mRNA biogenesis and is expressed ubiquitously. Studies in various model organisms have shown that the loss of function of the SMN gene leads to embryonic lethality. The human contains two genes encoding for SMN protein and in patients one of these is disrupted. It is thought the remaining low levels of protein produced by the second SMN gene do not suffice and result in the observed specific loss of lower motor neurons and muscle wasting. The early lethality in the animal mutants has made it difficult to understand why primarily these tissues are affected. We have isolated a Drosophila smn mutant. The fly alleles contain point mutations in smn similar to those found in SMA patients. We find that zygotic smn mutant animals show abnormal motor behavior and that smn gene activity is required in both neurons and muscle to alleviate this phenotype. Physiological experiments on the fly smn mutants show that excitatory post-synaptic currents are reduced while synaptic motor neuron boutons are disorganized, indicating defects at the neuromuscular junction. Clustering of a neurotransmitter receptor subunit in the muscle at the neuromuscular junction is severely reduced. This new Drosophila model for SMA thus proposes a functional role for SMN at the neuromuscular junction in the generation of neuromuscular defects.     

Human smooth pursuit during transient perturbations of predictable and unpredictable target movement

Summary The predictive component of human smooth pursuit was studied by perturbing sinusoidal target motion at unpredictable instants. The disturbances consisted of either a brief period of stabilization of the target on the fovea or a replacement of the sine by a ramp displacement for half a period. To minimize the effects of a possible change of the tracking strategy by the subject the transitions were masked and only the initial part of the response to the disturbance was analyzed. After stabilization on the fovea the eye oscillation continued at the frequency of the preceding target movement for about one half-cycle, whereupon the oscillation was rapidly damped. The mean unidirectional smooth eye acceleration was 70% of the mean unidirectional target acceleration prior to the stabilization. This suggests that during pursuit of a sinusoidal target movement about 75% of the oculomotor response is generated by predictive processes. When the sine was replaced by a ramp, starting at the velocity zero-crossing, the eye accelerated away from the target for ca. 180 ms irrespective of the frequency of prior tracking. In contrast, when the ramp started at the peak velocity of the sinusoidal target motion the eye accelerated away from the target for more than a quarter period. After foveal stabilization during pursuit of a pseudorandom stimulus, the eye continued to oscillate for less than one period at approximately the highest frequency present in the stimulus. The frequency characteristics of human smooth pursuit of predictable as well as unpredictable target motion were correctly simulated by a model, which derived its predictive properties from a lead element, tuned to the current frequency of the target motion.