Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors.

Mouse fibroblasts (3T3-L1 cells) accumulate detergent- and salt-insoluble aggregates of proteins conjugated to ubiquitin when incubated in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. These ubiquitin-protein conjugates co-fractionate with lysosomes on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. Both E-64 and ammonium chloride increase the intracellular concentration of free ubiquitin, but only E-64 leads to the formation of insoluble lysosomal ubiquitin-protein conjugates. The results are discussed in relation to the possible intracellular roles of ubiquitin conjugation.     

Gill monogenea of Rastrelliger spp. (Scombridae)

The following gill monogeneans are described, based on a survey of 240 Rastrelliger kanagurta, 12 R. faughni and 185 R. brachysoma (Scombridae) from many geographical areas: Eyelavera typica from R. kanagurta, R. faughni and R. brachysoma, Indomazocraes jagannath from R. kanagurta and R. faughni, Kuhnia sprostonae from R. kanagurta and R. brachysoma, and Kuhnia scombercolias from R. kanagurta and R. brachysoma. Eyelavera parukhini Lebedev, 1980 is synonymised with E. typica, Scomberocotyle eyela Unnithan, 1964 with Indomazocraes jagannath, Kuhnia microlepidotusi Gupta & Krishna, 1977 and K. kanagurta Mamaev & Parukhin, 1986 with K. sprostonae, K. arabica Mamaev & Parukhin, 1986 with K. scombercolias Nasir & Fuentes Zambrano, 1983. It is emphasized that populations of Monogenea from the same host species or genus in different geographical areas are likely to be conspecific, and should not be described as different species, if they differ only slightly from each other. Monogenea that differ from insufficiently described species in minor detail should not be described as new species unless material of the original species has been examined.     

N-15 in vivo NMR spectroscopic investigation of nitrogen deprived cell suspensions of the green alga Chlorella fusca

The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K¹⁵NO₃ after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected. Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids. The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo NMR spectroscopy can be applied to the investigation of N metabolism of the cells.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Biosynthesis of endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase

In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.