Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.     

Induction of PEP carboxylase and crassulacean acid metabolism by gibberellic acid in Mesembryanthemum crystallinum

The induction of Crassulacean acid metabolism in M:esembryanthemum crystallinum was investigated in response to foliar application of gibberellic acid (GA). After 5 weeks of treatment, GA-treated plants showed 1.7- to almost a 4-fold increase of phosphoenolpyruvate carboxylase (PEPcase) activity with a concomitant increase in acid metabolism when compared to control plants. Immunoblot analysis indicated an increase in the PEPcase protein similar to that of salt treatment while Rubisco did not show a similar rise. The results indicate that exogenously applied GA accelerates plant developmental expression of PEPcase and Crassulacean acid metabolism in M: crystallinum.     

The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme

The Azotobacter vinelandii genome encodes a family of seven secreted Ca(2+)-dependent epimerases (AlgE1--7) catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1--7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca(2+)-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G/MM and/or G/GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.     

Innate immunity and its evasion and suppression by hymenopteran endoparasitoids

Recent studies suggest that insects use pattern recognition molecules to distinguish prokaryotic pathogens and fungi from "self" structures. Less understood is how the innate immune system of insects recognizes endoparasitic Hymenoptera and other eukaryotic invaders as foreign. Here we discuss candidate recognition factors and the strategies used by parasitoids to overcome host defense responses. We suggest that host-parasitoid systems are important experimental models for studying how the innate immune system of insects recognizes foreign invaders that are phylogenetically more closely related to their hosts. The strategies used by parasitoids suggest that insects may employ "hidden-self" recognition molecules for attacking foreign objects intruding the open circulatory system. BioEssays 23:344-351, 2001.     

Alanine-scanning mutagenesis of plasmatocyte spreading peptide identifies critical residues for biological activity

Plasmatocyte spreading peptide (PSP) is a 23-amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. The structure of PSP consists of a disordered N terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bridge between Cys(7) and Cys(19), hydrophobic interactions, and a short beta-hairpin. Structural comparisons also indicate that the core region of PSP adopts an epidermal growth factor (EGF)-like fold very similar to the C-terminal subdomain of EGF-like module 5 of thrombomodulin. To identify residues important for plasmatocyte spreading activity, we bioassayed PSP mutants in which amino acids were either replaced with alanine or deleted. Within the well-defined core of PSP, alanine replacement of Cys(7) and Cys(19) (C7.19A) eliminated all activity. Alanine replacement of Arg(13) reduced activity approximately 1000-fold in comparison to wild-type PSP, whereas replacement of the other charged residues (Asp(16), Arg(18), Lys(20)) surrounding Cys(19) diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of wild-type PSP. The mutant PSP-(7-23) lacked the entire unstructured domain of PSP and was found to have no plasmatocyte spreading activity. Surprisingly, E1A and N2A had higher activity than wild-type PSP, but F3A had almost no activity. We thus concluded that the lack of activity for PSP-(7-23) was largely due to the critical importance of Phe(3). To determine whether reductions in activity correlated with alterations in tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants to wild-type PSP by NMR spectroscopy. As expected, the simultaneous replacement of Cys(7) and Cys(19) profoundly affected tertiary structure, but the R13A, R18A, and F3A mutants did not differ from wild-type PSP. Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are required for plasmatocyte-spreading activity.     

Long-distance seed dispersal in plant populations

Long-distance seed dispersal influences many key aspects of the biology of plants, including spread of invasive species, metapopulation dynamics, and diversity and dynamics in plant communities. However, because long-distance seed dispersal is inherently hard to measure, there are few data sets that characterize the tails of seed dispersal curves. This paper is structured around two lines of argument. First, we argue that long-distance seed dispersal is of critical importance and, hence, that we must collect better data from the tails of seed dispersal curves. To make the case for the importance of long-distance seed dispersal, we review existing data and models of long-distance seed dispersal, focusing on situations in which seeds that travel long distances have a critical impact (colonization of islands, Holocene migrations, response to global change, metapopulation biology). Second, we argue that genetic methods provide a broadly applicable way to monitor long-distance seed dispersal; to place this argument in context, we review genetic estimates of plant migration rates. At present, several promising genetic approaches for estimating long-distance seed dispersal are under active development, including assignment methods, likelihood methods, genealogical methods, and genealogical/demographic methods. We close the paper by discussing important but as yet largely unexplored areas for future research.     

Anaerobic Naphthalene Degradation by Microbial Pure Cultures under Nitrate-Reducing Conditions

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added ¹⁴C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the ¹⁴C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.