Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.     

Evaluation of biological availability and anti-arrhythmic effect of a new drug Phenytoinum "Polfa"]

Studies were performed in 15 patients with ventricular arrhythmia. During the first day, the patients received 1000 mg of a new micronised form of Phenytoinum "Polfa" or adequate dose of a foreign drug in 3 doses every 3 hours and subsequently during 10 days alternatively native or foreign drug in a daily dose 300 mg. Twenty-four EKG Holter monitoring and determination of serum drug level were carried out after a 10-day treatment; area under the curve (AUC) in one 8 h dose interval was determined. Studies have shown usefulness of a new form of Phenytoinum (Polfa). Blood serum drug levels near to the therapeutic ones were observed. Steady-state Phenytoinum concentration was 11.1 +/- 5.9 micrograms/ml and after foreign drug it was 11.7 +/- 6.1 micrograms/ml, AUC0-8 was 90.4 and 105.3 micrograms/ml/h respectively. In 9/15 patients (60%) Phenytoinum (Polfa) produced substantial improvement in the cardiac arrhythmia.     

Influence of sialic acids on the galactose-recognizing receptor of rat peritoneal macrophages

The interaction of the galactose-recognizing receptor from rat peritoneal macrophages with ligands containing terminal galactose residues, such as asialoorosomucoid, desialylated erythrocytes or lymphocytes, can be inhibited by free N-acetylneuraminic acid (Neu5Ac) and oligosaccharides or glycoproteins containing this sugar in terminal position. This effect of Neu5Ac on the receptor is specific. The other naturally occurring or most of synthetic neuraminic acid derivatives tested do not exhibit an equivalent inhibitory potency as Neu5Ac. Although free Neu5Ac inhibits 5-fold stronger (K50 = 0.2mM) than free galactose, clustering of Neu5Ac in oligosaccharides and glycoproteins does not lead to stronger inhibition, which is in contrast to galactose-containing ligands. A more branched (triantennary) sialooligosaccharide inhibits less than biantennary and unbranched sialooligosaccharides. This may be the reason, why complex sialic acid-containing ligands like native orosomucoid or blood cells are not bound and internalized by the macrophages. The dissociation of asialoorosomucoid from the receptor is slow under the influence of Neu5Ac and requires relatively high concentrations of this sugar, whereas the dissociation mediated by galactose is rapid and requires lower concentrations. An allosteric influence of Neu5Ac on the binding of galactose by the receptor is discussed.     

Characterization of a novel type of chain-terminator Gal beta 1-6Gal beta 1-4)GlcNAc in an oligosaccharide related to N-glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical-ionization mass spectrometry and 400-MHz 1H-NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N-acetyllactosamine-type glycan: Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3Man beta 1-4GlcNAc. The second is unusual with a terminal disaccharide Gal beta 1-6Gal, which had not yet been described for glycans of the N-acetyllactosamine type: Gal beta 1-6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6Man beta 1-4GlcNAc.     

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.     

Combined exposure to hydroelectric expansion,climate change and forest loss jeopardies amphibians in the Brazilian Amazon

Aim

Human‐driven impacts constantly threat amphibians, even in largely protected regions such as the Amazon. The Brazilian Amazon is home to a great diversity of amphibians, several of them currently threatened with extinction. We investigated how climate change, deforestation and establishment of hydroelectric dams could affect the geographic distribution of Amazonian amphibians by 2030 and midcentury.

Location

The Brazilian Amazon.

Methods

We overlapped the geographic distribution of 255 species with the location of hydroelectric dams, models of deforestation and climate change scenarios for the future.

Results

We found that nearly 67% of all species and 54% of species with high degree of endemism within the Legal Brazilian Amazon would lose habitats due to the hydroelectric overlapping. In addition, deforestation is also a potential threat to amphibians, but had a smaller impact compared to the likely changes in climate. The largest potential range loss would be caused by the likely increase in temperature. We found that five amphibian families would have at least half of the species with over 50% of potential distribution range within the Legal Brazilian Amazon limits threatened by climate change between 2030 and 2050.

Main conclusions

Amphibians in the Amazon are highly vulnerable to climate change, which may cause, directly or indirectly, deleterious biological changes for the group. Under modelled scenarios, the Brazilian Government needs to plan for the development of the Amazon prioritizing landscape changes of low environmental impact and economic development to ensure that such changes do not cause major impacts on amphibian species while reducing the emission of greenhouse gases.

     

Structure of the phosphopeptidomannans from flocculent and non-flocculent yeast Kluyveromyces lactis

After extraction from whole cells, and purification by gel filtration, the chemical composition and molecular mass estimation of the cell-wall phosphopeptidomannan (PPM) showed no significant difference respectively between flocculent, weakly, very weakly and non-flocculent Kluyveromyces lactis yeast strains. However, when PPMs were tested as ligands of a lectin, extracted from the flocculent strain, the PPM isolated from the flocculent and weakly flocculent strain were recognized to a higher degree than those isolated from the non and very weakly flocculent strains. Acetolysis of PPM extracted from the four strains produced five oligosaccharide fractions corresponding to mono-, di-, tri-, penta-and hexa-saccharides. The flocculent strain was characterised by a high content of di-and penta-saccharides. The ¹H NMR analysis of the oligosaccharides demonstrated that the flocculent strain contained equivalent levels of the two mannobioses: Man( 1 → 2)Man and Man( 1 → 3)Man and of the two mannotrioses Man( 1 → 2)Man( 1 → 2)Man and Man( 1 → 3)Man( 1 → 2)Man. In contrast, the non-flocculent and the very weakly flocculent strains contained a single type of mannobiose Man( 1 → 2)Man and one type of mannotriose Man( 1 → 2)Man( 1 → 2)Man.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

Plants adapted to warmer climate do not outperform regional plants during a natural heat wave

With ongoing climate change, many plant species may not be able to adapt rapidly enough, and some conservation experts are therefore considering to translocate warm‐adapted ecotypes to mitigate effects of climate warming. Although this strategy, called assisted migration, is intuitively plausible, most of the support comes from models, whereas experimental evidence is so far scarce. Here we present data on multiple ecotypes of six grassland species, which we grew in four common gardens in Germany during a natural heat wave, with temperatures 1.4–2.0°C higher than the long‐term means. In each garden we compared the performance of regional ecotypes with plants from a locality with long‐term summer temperatures similar to what the plants experienced during the summer heat wave. We found no difference in performance between regional and warm‐adapted plants in four of the six species. In two species, regional ecotypes even outperformed warm‐adapted plants, despite elevated temperatures, which suggests that translocating warm‐adapted ecotypes may not only lack the desired effect of increased performance but may even have negative consequences. Even if adaptation to climate plays a role, other factors involved in local adaptation, such as biotic interactions, may override it. Based on our results, we cannot advocate assisted migration as a universal tool to enhance the performance of local plant populations and communities during climate change.