Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand

Erythrina cristagalli agglutinin, a dimeric lectin [J. L. Iglesias, et al. (1982) Eur. J. Biochem.123, 247–252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-β-d-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (Δ? = 1.2 × 10³m^?1 cm^?1). A similar spectrum with a comparable value of Δ? was obtained with 4-methylumbelliferyl-N-acetyl-β-d-galactosaminide. Binding of methyl-α-d-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (Δ? = 2.8 × 10² M^?1 cm^?1) at 291.6 nm. Upon binding of N-dansyl-d-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-d-galactosamine was caused by a very favorable ΔS° of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-d-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(β → 4)GlcNAc(β1 → 2)Man, Gal(β1 → 4)GlcNAc(β1 → 6)Man, and Gal(β1 → 4)GlcNAc(β1 → 6)[Gal(β1 → 4)GlcNAc(β1 → 2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-d-galactosamine, methyl-α-d-galactoside, and lactose, ?ΔH° increased from 24 to 41 kJ mol^?1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 ± 1 kJ mol^?1). This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of ΔS° values which became more unfavorable in the above series (?23 to ?101 ± 4 J mol^?1 K^?1); the most negative value of ΔS° was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.     

Rhodospirillum salinarum sp. nov., a halophilic photosynthetic bacterium isolated from a Portuguese saltern

A new species of halophilic photosynthetic bacteria, Rhodospirillum salinarum, has been isolated and described. Its natural habitat are the terminal crystallization ponds of solar salt production plants. R. salinarum grows optimally at 42°C in the presence of 6–18% NaCl (w/v). Growth requirements are complex, yeast extract and peptone being required both for aerobic heterotrophic and for anaerobic phototrophic growth. Increasing concentrations of NaCl in the growth media did not give rise to any corresponding increase in intracellular concentrations of K⁺, Na⁺, polyalcohols or amino acids. Malate dehydrogenase from R. salinarum is not halophilic, being inhibited even at low concentrations of Na⁺ or K⁺. The GC mol % of DNA from R. salinarum is markedly higher than that for DNA from R. salexigens, the only previously described halophilic species of the genus Rhodospirillum.     

Continuous titration of difference absorption upon binding of 4-methylumbelliferyl glycosides to concanavalin A and peanut agglutinin

A continuous titration of absorption differences is described. Equal volumes of the titration fluid are dispensed from two micrometer-driven Hamilton gas-tight syringes into two 1 × 1 × 4.5-cm cuvettes. These are placed in the reference and sample beam. Each cuvette stopper is equipped with a capillary inlet connected to a syringe and with a minimotor for continuous stirring. Details of the stirring device are given. The delivered volumes of titration fluid are sufficiently reproducible to allow titration of absorption differences as a function of chromophore concentration. The usefulness of this approach is tested with the binding of 4-methylumbelliferyl α-d-mannopyranoside and concanavalin A as a well-characterized system. It is applied to the binding of similarly labeled anti-t disaccharide with the lectin from peanuts. With both lectins, the change in molecular extinction coefficient of the ligand and the association constant, valid for the entire protein saturation range, were obtained. The results are identical to those from other methods, including equilibrium dialysis.     

Bovine Brain Myelin Glycerophosphocholine Choline Phosphodiesterase is an Alkaline Lysosphingomyelinase of the eNPP-Family, Regulated by Lysosomal Sorting

Glycerophosphocholine choline phosphodiesterase (GPC-Cpde) is a glycosylphosphatidylinositol (GPI)-anchored alkaline hydrolase that is expressed in the brain and kidney. In brain the hydrolase is synthesized by the oligodendrocytes and expressed on the myelin membrane. There are two forms of brain GPC-Cpde, a membrane-linked (mGPC-Cpde) and a soluble (sGPC-Cpde). Here we report the characterisation sGPC-Cpde from bovine brain. The amino acid sequence was identical to ectonucleotide pyrophosphatase/phosphodiesterase 6 (eNPP6) precursor, lacking the N-terminal signal peptide region and a C-terminal stretch, suggesting that the hydrolase was solubilised by C-terminal proteolysis, releasing the GPI-anchor. sGPC-Cpde existed as two isoforms, a homodimer joined by a disulfide bridge linking C414 from each monomer, and a monomer resulting from proteolysis N-terminally to this disulfide bond. The only internal disulfide bridge, linking C142 and C154, stabilises the choline-binding pocket. sGPC-Cpde was specific for lysosphingomyelin, displaying 1 to 2 orders of magnitude higher catalytic activity than towards GPC and lysophosphatidylcholine, suggesting that GPC-Cpde may function in the sphingomyelin signaling, rather than in the homeostasis of acylglycerophosphocholine metabolites. The truncated high mannose and bisected hybrid type glycans linked to N118 and N341 of sGPC-Cpde is a hallmark of glycans in lysosomal glycoproteins, subjected to GlcNAc-1-phosphorylation en route through Golgi. Thus, sGPC-Cpde may originate from the lysosomes, suggesting that lysosomal sorting contributes to the level of mGPC-Cpde on the myelin membrane.     

Review: Pathogenesis of Helicobacter pylori infection

The original strategies developed by Helicobacter pylori to persistently colonise its host and to deregulate its cellular functions make this bacterium an outstanding model to study host‐pathogen interaction and the mechanisms responsible for bacterial‐induced carcinogenesis. During the last year, significant results were obtained on the role of bacterial factors essential for gastric colonisation such as spiral shape maintenance, orientation through chemotaxis and the formation of bacteria clonal population islands inside the gastric glands. Particularities of the H pylori cell surface, a structure important for immune escape, were demonstrated. New insights in the bacterial stress response revealed the importance of DNA methylation‐mediated regulation. Further findings were reported on H pylori components that mediate natural transformation and mechanisms of bacterial DNA horizontal transfer which maintain a high level of H pylori genetic variability. Within‐host evolution was found to be niche‐specific and probably associated with physiological differences between the antral and oxyntic gastric mucosa. In addition, with the progress of CryoEM, high‐resolution structures of the major virulence factors, VacA and CagT4SS, were obtained. The use of gastric organoid models fostered research revealing, preferential accumulation of bacteria at the site of injury during infection. Several studies further characterised the role of CagA in the oncogenic properties of H pylori, identifying the activation of novel CagA‐dependent pathways, leading to the promotion of genetic instabilities, epithelial‐to‐mesenchymal transition and finally carcinogenesis. Recent studies also highlight that microRNA‐mediated regulation and epigenetic modifications, through DNA methylation, are key events in the H pylori‐induced tumorigenesis process.     

Root engineering in maize by increasing cytokinin degradation causes enhanced root growth and leaf mineral enrichment

Plant Molecular Biology - Root-specific expression of a cytokinin-degrading CKX gene in maize roots causes formation of a larger root system leading to higher element content in shoot organs. The...     

The geometry and genetics of hybridization

When divergent populations form hybrids, hybrid fitness can vary with genome composition, current environmental conditions, and the divergence history of the populations. We develop analytical predictions for hybrid fitness, which incorporate all three factors. The predictions are based on Fisher's geometric model, and apply to a wide range of population genetic parameter regimes and divergence conditions, including allopatry and parapatry, local adaptation, and drift. Results show that hybrid fitness can be decomposed into intrinsic effects of admixture and heterozygosity, and extrinsic effects of the (local) adaptedness of the parental lines. Effect sizes are determined by a handful of geometric distances, which have a simple biological interpretation. These distances also reflect the mode and amount of divergence, such that there is convergence toward a characteristic pattern of intrinsic isolation. We next connect our results to the quantitative genetics of line crosses in variable or patchy environments. This means that the geometrical distances can be estimated from cross data, and provides a simple interpretation of the “composite effects.” Finally, we develop extensions to the model, involving selectively induced disequilibria, and variable phenotypic dominance. The geometry of fitness landscapes provides a unifying framework for understanding speciation, and wider patterns of hybrid fitness.