Equilibrium and kinetic studies of the interactions of a porphyrin with low-density lipoproteins

Low-density lipoproteins (LDL) play a key role in the delivery of photosensitizers to tumor cells in photodynamic therapy. The interaction of deuteroporphyrin, an amphiphilic porphyrin, with LDL is examined at equilibrium and the kinetics of association/dissociation are determined by stopped-flow. Changes in apoprotein and porphyrin fluorescence suggest two classes of bound porphyrins. The first class, characterized by tryptophan fluorescence quenching, involves four well-defined sites. The affinity constant per site is 8.75 x 10(7) M(-1) (cumulative affinity 3.5 x 10(8) M(-1)). The second class corresponds to the incorporation of up to 50 molecules into the outer lipidic layer of LDL with an affinity constant of 2 x 10(8) M(-1). Stopped-flow experiments involving direct LDL porphyrin mixing or porphyrin transfer from preloaded LDL to albumin provide kinetic characterization of the two classes. The rate constants for dissociation of the first and second classes are 5.8 and 15 s(-1); the association rate constants are 5 x 10(8) M(-1) s(-1) per site and 3 x 10(9) M(-1) s(-1), respectively. Both fluorescence and kinetic analysis indicate that the first class involves regions at the boundary between lipids and the apoprotein. The kinetics of porphyrin-LDL interactions indicates that changes in the distribution of photosensitizers among various carriers could be very sensitive to the specific tumor microenvironment.     

A two-protein strategy for the functional loading of a cellular replicative DNA helicase

The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.     

Molecular cloning and biological activity of alpha-, beta-, and gamma-megaspermin,three elicitins secreted by Phytophthora megasperma H20

We report on the molecular cloning of the Phytophthora megasperma H20 (PmH20) glycoprotein shown previously as an inducer of the hypersensitive response, of localized acquired resistance and of systemic acquired resistance in tobacco (Nicotiana tabacum), and of the PmH20 alpha- and beta-megaspermin, two elicitins of class I-A and I-B, respectively. The structure of the glycoprotein shows a signal peptide of 20 amino acids followed by the typical elicitin 98-amino acid-long domain and a 77-amino acid-long C-terminal domain carrying an O-glycosylated moiety. The molecular mass deduced from the translated cDNA sequence is 14,920 and 18,676 D as determined by mass spectrometry. This structure together with multiple sequence alignments and phylogenetic analyses indicate that the glycoprotein belongs to class III elicitins. It is the first class III elicitin protein characterized, which we named gamma-megaspermin. We compared the biological activity of the three PmH20 elicitins when applied to tobacco cv Samsun NN plants. Although alpha- and gamma-megaspermin were similarly active, beta-megaspermin was the most active in inducing the hypersensitive response and localized acquired resistance, which was assessed by measuring the levels of acidic and basic pathogenesis-related proteins and of the antioxidant phytoalexin scopoletin. The three elicitins induced similar levels of systemic acquired resistance measured as the expression of acidic PR proteins and is increased resistance to challenge tobacco mosaic virus infection.     

Aluminum speciation studies in biological fluids. Part 9. A quantitative investigation of aluminum(III)-glutamate complex equilibria and their potential implications for aluminum metabolism and toxicity

As a nonessential element, aluminum is likely to be toxic both at low usual dietary levels in the long run (chronic toxicity) and at high therapeutic levels in shorter periods of time (acute toxicity). In both situations, aluminum toxicity is a direct function of aluminum bioavailability, which is itself dependent on Al(3+) solubility and charge neutralization. Dietary acids, by their intrinsic acidity and coordinating capacity, can extend the pH range, thus the section of the gastrointestinal tract, within which the Al(3+) ion remains soluble, and also help Al(3+) diffusion across the intestinal epithelium through the formation of neutral complex species. The present work examines the impact of glutamic acid, an essential amino acid also widely used in industrial food and drinks, on aluminum speciation in the gastrointestinal tract and blood plasma. Complex formation between the Al(3+) ion and glutamate has first been investigated through potentiometric titrations, complex stoichiometries being then checked by ESI mass spectrometry and NMR measurements. A series of mono- and polynuclear species has been characterized, whose influence on aluminum distribution in vivo has been assessed by computer simulation. The capacity of glutamate to maintain Al(3+) ions in solution under normal dietary conditions is predicted to be intermediate between glycine-like amino acids and succinate on the one hand, and tartrate and malate on the other hand, its Al(3+) neutralization effect being similar to that of succinate, tartrate and malate. These results, which point to a potential aggravating role of glutamate on aluminum gastrointestinal absorption, substantiate recent observations made on rats. In spite of the moderate effect expected from glutamate on aluminum bioavailability under most aluminum-based therapies investigated, attention is therefore called to the risk of glutamic acid ingestion simultaneously to any aluminum therapeutic form. Incidentally, the former implication of 'the' aluminum glutamate complex in the transfer of aluminum through the blood-brain barrier of aluminum loaded rats may effectively be attributed to one of the species characterized here, but is of no significance at all to aluminum contamination in humans, even at most extreme levels.     

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a (13)C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.     

Lateralization defects and ciliary dyskinesia: lessons from algae

Flagella and cilia are two very similar organelles that "beat" to move cells and to propel fluid over tissues. They are highly conserved, being found in organisms ranging from prokaryotes to plant and animal eukaryotes. In humans, cilia are present in almost every organ, and several human conditions involve dysfunctional cilia; for example, lateralization defects, where the positions of organs are reversed, and primary ciliary dyskinesia, a rare condition where patients suffer from recurrent respiratory infections. In this article, we will discuss how information gained from studies on algae has aided research into these human diseases. These studies found a variety of functions that was previously unsuspected, renewing interest in cilia.     

Mek2 is dispensable for mouse growth and development

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.