Anti-stress activity of Propionibacterium freudenreichii: identification of a reactivative protein

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.     

Morphogenesis of post-Golgi transport carriers

The trans-Golgi network (TGN) is one of the main, if not the main, sorting stations in the process of intracellular protein trafficking. It is therefore of central importance to understand how the key players in the TGN-based sorting and delivery process, the post-Golgi carriers (PGCs), form and function. Over the last few years, modern morphological approaches have generated new insights into the questions of PGC biogenesis, structure and dynamics. Here, we present a view by which the “lifecycle” of a PGC consists of several distinct stages: the formation of TGN tubular export domains (where different cargoes are segregated from each other and from the Golgi enzymes); the docking of these tubular domains onto molecular motors and their extrusion towards the cell periphery along microtubules; the fission of the forming PGC from the donor membrane; and the delivery of the newly formed PGC to its specific acceptor organelle. It is now important to add the many molecular machineries that have been described as operating at the TGN to this “morphofunctional map” of the TGN export process.     

Anti-alpha-galactosyl antibodies recognizing epitopes terminating with alpha1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.     

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 andD-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: Role of Mg2+ ions

Agonists modulation of Mg²⁺-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg²⁺ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with higher and lower apparent affinity states, are extrapolated. In the presence ofD-Ala²-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E₂ increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg²⁺-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

Further expansion of the genus Cercopagis (Crustacea,Branchiopoda, Onychopoda) in the Baltic Sea,with notes on the taxa present and their ecology

The onychopod cladoceran Cercopagis that recently invaded the Baltic Sea is reported from new zones of the northern Baltic proper. Because of successful survival and an expanding distribution range, the addition of Cercopagis to the Baltic fauna is considered to be permanent. What has previously been cited as Cercopagis pengoi encompasses the morphology of several other species, subspecies and forms. Either a number of morphologically similar species is present, or there is a number of spurious species in Cercopagis. The last hypothesis is favoured. The spatial distribution pattern of Cercopagis, as well as that of total zooplankton, was correlated with depth. Deep (>100 m) and shallow (<10 m) stations had significantly lower abundance than stations of intermediate depth (<100 m). An overview of the distribution of C. pengoi group in fresh and brackish waters suggests a high tolerance to environmental factors, but with differences among taxa. Due to this ecological flexibility, the colonization of the Baltic is not unexpected. Increasing salinity may restrict dispersal of cercopagids to the southern areas of the Baltic and to the North Sea, but inland lakes (e.g. in Sweden) present an ecological profile suitable for colonization.     

Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes

F. VILLANI, G. SALZANO, E. SORRENTINO, O. PEPE, P. MARINO AND S. COPPOLA. 1993. Enterococcus faecalis 226, isolated from natural whey cultures utilized as starters in the manufacture of mozzarella cheese from water-buffalo milk, produces a bacteriocin designated enterocin 226NWC. The bacteriocin was isolated from culture supernatant fluids of the producer strain and was active against strains of the same species and Listeria monocytogenes, but not against useful lactic acid bacteria. Enterocin 226NWC is a protein with an apparent molecular weight of about 5800; it is relatively heat-stable and has a bactericidal mode of action. Listeria monocytogenes, growing in the presence of the enterocin 226NWC producer strain in broth and in reconstituted skim milk, was inhibited.