Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

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     

Knowledge-based modeling of the D-lactate dehydrogenase three-dimensional structure

A three-dimensional structure of the NAD-dependent D -lactate dehydrogenase of Lactobacillus bulgaricus is modeled using the structure of the formate dehydrogenase of Pseudomonas sp. as template. Both sequences share only 22% of identical residues. Regions for knowledge-based modeling are defined from the structurally conserved regions predicted by multiple alignment of a set of related protein sequences with low homology. The model of the D -LDH subunit shows, as for the formate dehydrogenase, an α/β structure, with a catalytic domain and a coenzyme binding domain. It points out the catalytic histidine (His-296) and supports the hypothetical catalytic mechanism. It also suggests that the other residues involved in the active site are Arg-235, possibly involved in the binding of the carboxyl group of the pyruvate, and Phe-299, a candidate for stabilizing the methyl group of the substrate. © 1995 Wiley-Liss, Inc.     

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.     

Synthesis of RNA I by the RNA polymerase from Micrococcus luteus on the Escherichia coli plasmid pBR322

A mutant strain of Bacillus megaterium, arising spontaneously and resistant to micrococcin , possesses ribosomes which contain an altered form of protein BM-L11 (the homologue of Escherichia coli protein L11). Reconstitution analysis has revealed that the alteration to protein BM-L11 is the sole cause of resistance in this strain.     

Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.