Analysis of Congo red-induced changes in the cell surface and macrocolony structure of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Adsorption of the vital dye Congo red suppresses swarming of Azospirillum brasilense in a semiliquid medium, and the bacteria become able to spread with the formation of microcolonies. By using direct and stereoscopic light microscopy, the patterns of the front of Azospirillum spreading in a semiliquid medium containing the dye were analyzed. It was found that in a medium with Congo red, small motile colonies were formed among the individual cells, and once formed, they left the boundaries of the swarming front. The microcolonies produced by azospirilla in the presence of the dye were ordered bacterial structures, rather than random cell aggregates. Transmission electron microscopy revealed that the cells grown without the dye had polar flagella, whereas the cells from the medium with Congo red had no flagella and were covered with a layer of fibrillike material. Immunochemical data for the cell surface changes resulting from interaction with the dye make it possible to consider Azospirillum lipopolysaccharide as a probable Congo red receptor.     

Three-Dimensional Organization of Polyribosomes–A Modern Approach

Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed tworow forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.     

Action of ω-conotoxin on calcium currents of rat pituitary GH3 cells

The effect of -conotoxin (-CgTX) on calcium currents of rat pituitary GH3 cells was studied by the voltage clamp method on a whole cell, under tight junction conditions. Two different components of the inward calcium current were observed in a solution containing 15 mM Ca²⁺. The first was activated with a holding potential of –80 mV and by testing pulses more positive than –55 mV. A shift of holding potential to –40 mV led to steady-state inactivation of this low-threshold component of the current. -CgTX at the initial moment after its application had an activating action on both components of the calcium current: low-threshold and high-threshold, but the increase in the first was much greater. In the present experiments the currents increased as early as 30 sec after replacement of the external solution; later the drop of current took place with temporal parameters characteristic of the spontaneous current drop in the control solution during cell dialysis. Incubation of the cells in growth medium containing 5 µM -CgTX for 2 h led to an increase in the density of both types of calcium currents in the GH3 cells, which was reduced after incubation for 2 h in the same medium. Thus -CgTX was found to have an activating action on calcium currents of GH3 cells at the initial moment after application of the toxin. The absence of a marked blocking action of -CgTX on the calcium currents of the test cells confirms the high tissue specificity of action of the toxin as a blocker of high-threshold calcium channels in the nerve cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 199–205, March–April, 1991.     

Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus

Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A. The diffraction patterns extend to better than 20 A.     

Isolation and characterisation of chitosanase from Bacillus sp. 739 strain

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 has been determined. Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively. Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h. With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

The Chitinolytic Activity of Bacillus Cohn Bacteria Antagonistic to Phytopathogenic Fungi

Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, -1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r _{x , y} = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Protein and peptide factors from Bacillus sp.739 inhibit the growth of phytopathogenic fungi

Thermolabile peptides inhibiting the growth of Helminthosporium sativum, a facultative phytopathogen, have been isolated from the low-molecular-weight fraction of extracellular metabolites of the strain Bacillus sp. 739. Paper chromatography of the fraction, followed by bioautography, revealed the presence of three components exhibiting antifungal activity. These components were separated by gel chromatography on Toyopearl HW-40. SDS-PAGE (the Laemmli procedure) demonstrated that only one component was a protein (MW, approximately 14 kDa). The other two substances were polypeptides with molecular weights less than 6 kDa each. The protein factor inhibited the growth of H. sativum with a minimum effective concentration of 0.1 to 0.2 mg/ml.