Antimicrobial activity of lysozyme isoforms: Key molecular features

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry‐heating increasing lysozyme activity. Dry‐heated lysozyme (DH‐L) is a mixture of isoforms (isoaspartyl, native‐like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH‐L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH‐L against E. coli , indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.     

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.     

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Background

Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.

Objective

The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.

Methods

Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.

Results

Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.

Conclusions & Clinical Relevance

DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.     

Amphiphilic Macromolecules on Cell Membranes: From Protective Layers to Controlled Permeabilization

Antimicrobial and cell-penetrating peptides have inspired developments of abiotic membrane-active polymers that can coat, penetrate, or break lipid bilayers in model systems. Application to cell cultures is more recent, but remarkable bioactivities are already reported. Synthetic polymer chains were tailored to achieve (i) high biocide efficiencies, and selectivity for bacteria (Gram-positive/Gram-negative or bacterial/mammalian membranes), (ii) stable and mild encapsulation of viable isolated cells to escape immune systems, (iii) pH-, temperature-, or light-triggered interaction with cells. This review illustrates these recent achievements highlighting the use of abiotic polymers, and compares the major structural determinants that control efficiency of polymers and peptides. Charge density, sp. of cationic and guanidinium side groups, and hydrophobicity (including polarity of stimuli-responsive moieties) guide the design of new copolymers for the handling of cell membranes. While polycationic chains are generally used as biocidal or hemolytic agents, anionic amphiphilic polymers, including Amphipols, are particularly prone to mild permeabilization and/or intracell delivery.     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

Differential contribution of C-terminal regions of dermorphin and dermenkephalin to opioid-sites selection and binding potency

Dermorphin and dermenkephalin are D-aminoacid containing peptides generated from processing of the plurifonctional biosynthetic precursor pro-dermorphin. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM) and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), are among the most selective and potent agonists described respectively for the mu- and delta-opioid receptors. In order to identify determinants of selectivity and high-affinity receptor binding of dermorphin and dermenkephalin, a series of analogs was investigated for their affinity at the mu- and delta-receptors in the brain. The tetrapeptide amino end of both DRM and DREK were found to display high affinity and selectivity towards mu-receptors. Substitution of the C-terminal tripeptide of DREK with that of DRM reversed the receptor selectivity of DREK from delta to mu. Replacement of the C-terminal tripeptide of DRM with the C-terminal counterpart of DREK shifted the selectivity of DRM from mu to delta. These data emphasize the critical contribution of the carboxy end of DREK to delta-selectivity. They further suggest that the potent mu-address lying in the N terminus of DREK is overwhelmed by the powerful delta-directing ability of the carboxy end. Unlike DREK, the C-terminus of DRM is not involved in opioid receptor sites selection but is important insofar as it serves to stabilize interactions of DRM with the mu-receptor binding site.     

Molecular determinants of receptor affinity and selectivity of the natural delta-opioid agonist, dermenkephalin

Processing of the polyprotein precursor pro-dermorphin generates two distantly related D-amino acid-containing peptides, dermorphin and dermenkephalin, which are among the most selective high affinity agonists described, respectively, for the mu- and delta-opioid receptors. Dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, is a linear, potentially flexible peptide devoid of structural homology with either enkephalins, endorphins, or dynorphins and, as such, represents a useful tool for identifying determinants of high affinity and selective binding of opioids to the delta-receptor. A series of selected dermenkephalin analogs and homologs was investigated for affinity at the mu- and delta-sites in the brain. Whereas dermenkephalin has high affinity and specificity for the delta-opioid receptors, its tetrapeptide amino end, dermenkephalin-[1-4]-NH2 binds almost exclusively at the mu-receptors. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, is only marginally more selective for the u-sites than is dermenkephalin-[1-4]-NH2. Using dermorphin-dermenkephalin peptide hybrids and C-terminal deletion analogs of dermenkephalin, we showed the critical role that the C-terminal residues Met6 and Asp7 play in specifying correct addressing of dermenkephalin toward delta-receptors. The potent mu-deteminant located within the amino end of dermenkephalin is over-whelmed by the powerful delta-directing ability of the carboxy end. The negatively charged side chain of Asp7 makes a significant contribution to the delta-addressing ability of the C-terminal region, a finding consistent with Schwyzer's membrane selection model (Schwyzer, R. (1986) Biochemistry 25, 6335-6342). The Leu residue in position 5 and D-configuration about the alpha-carbon of Met2 were found to be of crucial importance for high affinity binding to delta-receptors. Whereas the Met residue in position 6 in dermenkephalin could safely be oxidized or replaced with D-Met, oxidation of Met2 led to deleterious effects, this analog being 1/100 as potent as dermenkephalin at delta-sites. Overall, the data collected demonstrate that highest levels of selectivity and affinity for the delta-opioid receptors can be achieved with small-sized, potentially flexible, linear peptides and further support the model according to which, in addition to optimum accommodation at the receptor, selection for delta-receptors is reduced by the effective positive charge of the molecule. Dermenkephalin may provide a starting point for the design of agonists and antagonists with nearly total specificity for the delta-sites. Such pharmacological agents could be used to explore the ill-defined physiological role and behavioral actions conveyed by delta-opioid receptors.