Effects of UV-B radiation and nitrogen starvation on enzyme activities in isolated plasma membranes of Euglena gracilis

Circadian rhythms are characteristic of many physiological and biochemical processes in the freshwater flagellate Euglena gracilis. Earlier, we found that the rhythms of photosynthesis, phototaxis and cell shape followed the same pattern in control organisms, but were differently affected by stress such as UV-B irradiation and nitrogen deficiency. Here we extend our studies to use isolated plasma membranes to characterize the rhythms of some plasma membrane-bound enzymes. Also, we wanted to see whether stress-induced changes of these rhythms could be detected at the subcellular level and possibly be coupled to the changes seen in photosynthesis, phototaxis and cell shape. The isolation of plasma membranes using aqueous polymer two-phase partitioning was successful, as judged by the large enrichment of the plasma membrane-marker 5′-nucleotidase, and the difference in the polypeptide pattern compared with the microsomal fraction from which it was prepared. Two other enzymes were analyzed, K⁺, Mg²⁺-ATPase, and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV-B radiation by ca 30–50%, compared with the control cultures. On the other hand, nitrogen deficiency not only reduced the activity of the K⁺.Mg²⁺-ATPase but also increased the activities of the 5′-nucleotidase and adenylyl cyclase. The different treatments also resulted in differences in polypeptide pattern, e.g., a polypeptide around 30 kDa seemed to be specific to plasma membranes of nitrogen-deficient cultures and one at 39 kDa for the UV-B radiated ones. All three enzymes showed diurnal rhythms that were affected by UV-B radiation. The peak in the rhythm of the ATPase was shifted by UV-B radiation, the rhythm of the 5′-nucleotidase nearly eliminated. The first peak of adenylyl cyclase activity was delayed, so that it looked more like a broad peak between 2 and 11 h after the onset of light. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV-B irradiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.     

Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole     

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt nucleotide(s) - PAGE polyacrylamide gel electrophoresis     

The ant’s path integration system: a neural architecture

 A model is developed by which path integration as observed in many animal species could be implemented neurobiologically. The proposed architecture is able to describe the navigation behaviour of Cataglyphis ants, and that of other social insects, at the level of interacting neurons. The basic idea of this architecture is the concept of activity patterns travelling along neural chains. Although experimental evidence has yet to be provided, this concept seems biologically plausible and not limited to the navigation problem. Neural chains are able to represent variables by activity patterns with high accuracy and temporal stability. Moreover, they are able to integrate incremental signals with high precision. Cyclical chains of neurons show superior performance as soon as cyclical variables are to be represented and integrated. Finally, representation of cyclical variables by travelling activity peaks allows simple approximations of goniometric functions as they are used in path integration systems. Received: 15 November 1994/Accepted in revised form: 30 May 1995     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Catecholamine-synthesizing enzymes in the rat stomach

Local production of catecholamines in the stomach of the rat was studied by immunohistochemical demonstration of tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), the enzymes catalyzing the formation of dopamine, noradrenaline and adrenaline, respectively. A rich innervation of TH- and DBH-immunoreactive nerve fibers was seen in the muscular layers and the myenteric plexus, in the submucosa and in the walls of submucosal blood vessels and in the lamina propria at the base of the epithelial layer. In addition, TH-, but not DBH-immunoreactive nerve fiber networks surrounding ganglion cells in the myenteric plexus were frequently observed, indicating dopaminergic preganglionic innervation of the myenteric plexus. In the oxyntic epithelium, single TH- and DBH-immunoreactive fibers extended in the strands of lamina propria as far as the middle portion of the gastric glands. A small population of single angulate cells in the oxyntic epithelium showed TH-, but not DBH-immunoreactivity. No specific PNMT immunoreactivity was observed.     

Culture of chondrocytes in medium supplemented with fetal calf serum or a serum substitute: Ultroser G

Fetal calf serum and a serum substitute, Ultroser G, were compared for their effects on the growth curves, clonal growth and cell cycle progression of rabbit chondrocytes in primary culture and during at least three cell passages and included a screen for the maintenance of cartilage-like differentiation i.e. the presence of type II collagen. Proliferation was also compared with another serum substitute, Nu-Serum. Ultroser G is shown to be equivalent to fetal calf serum as far as chondrocyte proliferation is concerned, clonal growth is improved and biosynthesis of type II collagen is maintained in primary culture.     

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

Total and free plasma concentrations of progesterone, cortisol and oestradiol-17 beta during pregnancy, parturition and early lactation in sows

The total (bound plus free) concentrations of progesterone, 20 alpha-dihydroprogesterone, oestradiol-17 beta and cortisol were determined in the plasma of sows at three stages during pregnancy and more intensively from 5 days pre-partum to 5 days post-partum. The free fractions of progesterone, oestradiol-17 beta and cortisol were measured in the same samples by a rate dialysis method. Up to day 110 of gestation, the amounts of free hormone in plasma did not fluctuate independently of their total concentrations. During farrowing, the total and free concentrations of progesterone and cortisol varied independently of each other, whereas total and free oestradiol-17 beta declined simultaneously. The initiation of parturition was associated with a decrease in circulating total progesterone, and was accentuated by a decrease in the free fraction (P less than 0.005) so that its active free concentration was only 20% of its day 1 pre-partum value. Total and free cortisol concentrations rose rapidly during labour so that at 12-18 h after birth of the first piglet 30% of that cortisol in maternal plasma was free hormone.