Valorisation of softwood bark through extraction of utilizable chemicals. A review.

Softwood bark is an important source for producing chemicals and materials as well as bioenergy. Extraction is regarded as a key technology for obtaining chemicals in general, and valorizing bark as a source of such chemicals in particular. In this paper, properties of 237 compounds identified in various studies dealing with extraction of softwood bark were described. Finally, some challenges and perspectives on the production of chemicals from bark are discussed.

Synthesis and biological evaluation of new simple indolic non peptidic HIV Protease inhibitors: the effect of different substitution patterns.

New structurally simple indolic non peptidic HIV Protease inhibitors were synthesized from (S)-glycidol by regioselective methods. Following the concept of targeting the protein backbone, different substitution patterns were introduced onto the common stereodefined isopropanolamine core modifying the type of functional group on the indole, the position of the functional group on the indole and the type of the nitrogen containing group (sulfonamides or perhydroisoquinoline), alternatively. The systematic study on in vitro inhibition activity of such compounds confirmed the general beneficial effect of the 5-indolyl substituents in presence of arylsulfonamide moieties, which furnished activities in the micromolar range. Preliminary docking analysis allowed to identify several key features of the binding mode of such compounds to the protease.

Molecular aspects of a novel HLA-A*02 allele (A*0297): the first HLA class I allele mutated at codon 232.

We describe a new HLA-A*02 allele, identified in a cord blood unit and in her mother. Nucleotide sequence analysis showed the presence of a new HLA-A*02 allele identical to HLA-A*02010101 except for a non-synonymous nucleotide exchange in exon 4 modifying codon 232 from GAG (Glu) to GAC (Asp). No other human leucocyte antigen class I allele sequenced so far shows this triplet at codon 232. The amino acid exchange affects a position that is part of the membrane proximal domain of class I major histocompatibility complex (MHC), designated alpha 3, and involved in the interaction with CD8 molecule. Using molecular modelling approach, the interactions between different subunits of the native and mutated forms of MHC-I resulted in relevant changes.

De novo design of potent antimicrobial peptides.

Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic beta-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed.

Molecular dynamics study on lipid A from Escherichia coli: insights into its mechanism of biological action.

Structural properties of the Escherichia coli lipid A moiety were analysed by means of molecular mechanics and molecular dynamics simulations and compared to synthetic monophospho and dephospho analogues with different biological activities in the Limulus assay. The conformation of glucosamine disaccharide headgroup, order and packing of fatty acid chains, solvation of phosphate groups, coordination by water molecules, sodium counterions and models of cationic amino acid side chains were described in terms of mean values, mean residence times, radial distribution functions, coordination numbers, solvation and interaction energies. Solvation and polar interactions of the phosphate groups were correlated to known biological activities the lipid A variants. The observed relationship between the biological effect and the number and position of the phosphate groups were explained with the help of simple mechanistic models of lipid A action. The possible mechanism of action involving specific binding of lipid A disaccharide headgroup to cationic residues of a receptor model was compared with an alternative mechanism, which assumes a relationship between the ability to adopt non-lamellar supramolecular structures and the biological activity. Conclusions are drawn about the probable mode of lipid A action. Implications for rational drug design of endotoxin-neutralising agents are discussed.

Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action.

Lipid A moiety has been identified as the bioactive component of bacterial endotoxins (lipopolysaccharides). However, the molecular mechanism of biological activity of lipid A is still not fully understood. This paper contributes to understanding of the molecular mechanism of action of bacterial endotoxins by comparing molecular modelling results for two possible mechanisms with the underlying experimental data. Mechanisms of action involving specific binding of lipid A to a protein receptor as well as nonspecific intercalation into phospholipid membrane of a host cell were modelled and analysed. As the cellular receptor for endotoxin has not been identified, a model of a peptidic pseudoreceptor was proposed, based on molecular structure, symmetry of the lipid A moiety and the observed character of endotoxin-binding sites in proteins. We have studied the monomeric form of lipid A from Escherichia coli and its seven synthetic analogues with varying numbers of phosphate groups and correlated them with known biological activities determined by the Limulus assay. Gibbs free energies associated with the interaction of lipid A with the pseudoreceptor model and intercalation into phospholipid membrane calculated by molecular mechanics and molecular dynamics methods were used to compare the two possible mechanisms of action. The results suggest that specific binding of lipid A analogues to the peptidic pseudoreceptor carrying an amphipathic cationic binding pattern BHPHB (B, basic; H, hydrophobic; P, polar residue, respectively) is energetically more favourable than intercalation into the phospholipid membrane. In addition, binding affinities of lipid A analogues to the best minimum binding sequence KFSFK of the pseudoreceptor correlated with the experimental Limulus activity parameter. This correlation enabled us to rationalize the observed relationship between the number and position of the phosphate groups in the lipid A moiety and its biological activity in terms of specific ligand-receptor interactions. If lipid A-receptor interaction involves formation of phosphate-ammonium ion-pair(s) with cationic amino-acid residues, the specific mechanism of action was fully consistent with the underlying experimental data. As a consequence, recognition of lipid A variants by an amphipathic binding sequence BHPHB of a host-cell protein receptor might represent the initial and/or rate-determining molecular event of the mechanism of action of lipid A (or endotoxin). The insight into the molecular mechanism of action and the structure of the lipid A-binding pattern have potential implications for rational drug design strategies of endotoxin-neutralizing agents or binding factors.

Molecular dynamics study on the conformational stability of laminaran oligomers in various solvents.

Solution conformations of the (1-->3)-beta-D-glucan laminaran have been investigated by means of molecular mechanics and molecular dynamics simulations in three solvents: water, dimethylformamide, and dimethyl sulfoxide. Conformational analysis of solvated laminarabiose was carried out to study the effect of specific solute-solvent interactions upon reactive hydroxyl groups. Dynamic trajectories of solvated laminarabiose have been interpreted in terms of average glycosidic-linkage conformation, hydrogen-bonding pattern, and coordination by solvent molecules. The analysis of radial distribution functions, coordination functions, mean residency times, and time correlation functions derived from the simulation trajectories furnished the necessary insight. The results have been used to assess the steric accessibility of laminaran-C4-OH and -C6-OH hydroxyl groups in the considered solvents and to rationalize corresponding earlier experimental observations. The experiments demonstrated that reaction equilibrium during chemical modification of laminaran oligomers with 2,2-dimethoxypropane involving the laminaran-C4-OH and -C6-OH hydroxyl groups significantly depended on the solvent used.

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Rational design of inhibitors for drug-resistant HIV-1 aspartic protease mutants.

This report describes a method for the assessment of inhibitor binding affinities to wild type HIV-1 aspartic protease and to its drug-resistant mutant forms. We have elaborated a refined method for molecular modeling of the 3D structures of mutant enzymes and enzyme-inhibitor complexes based on the crystal structure of the wild type form, which employs a full thermodynamic cycle. Model complexes of four HIV-1 aspartic protease mutants with ten analogs of the A77003 inhibitor were considered. Predictions of inhibition efficiency, resistance potential, and hydrophilicity of the redesigned A77003 analogs were obtained by employing molecular mechanics for the evaluation of enzyme-inhibitor complexation energy and the polarizable continuum model for the estimation of solvent effects. Simple qualitative indicators for structural modifications aimed at overcoming the emergence of HIV resistance to protease inhibitors and at increasing the bioavailability of pseudopeptide inhibitors are examined. A semi-quantitative method for the description of enzyme-ligand binding and its implications for the rational design of inhibitors with higher binding affinity towards emerging HIV PR mutants is presented.

Ab initio studies of aromatic-aromatic and aromatic-polar interactions in the binding of substrate and inhibitor to dihydrofolate reductase.

Aromatic-aromatic and aromatic-polar interactions are investigated by performing ab initio Hartree-Fock calculations. Binding energies and optimum distances between subsystems are obtained. It is found that the binding energy between two benzene rings is of 3.1 kcal/mol when correlation effects are included, while the serine aromatic complexes energies of binding range from 1.9 to 3.1 kcal/mol.

Theoretical study of N-nitrosoureas and mechanism of their carcinogenic effect.

A mechanistic study into the carcinogenic action of N-nitrosoureas (NU) was carried out with the aid of the semiempirical MINDO/3 method. The proposed reaction pathways of NU biodegradation mechanisms were examined and the most probable one was identified on the ground of theoretical considerations. The calculations of reaction enthalpies confirmed NU as SN1 reagents. The reactivity of a series of NU molecules (both isolated and under the solvent influence) were studied with respect to the detection of possible determinants of the relative carcinogenic potency. The correlations revealed a key function of the nitrosogroup and of the N3-C7 fragment in the decomposition process. The role of the transport properties and lipophilicity of parent NU molecules in the initial steps of the mechanism of carcinogenic effects was demonstrated as well.

Theoretical QSAR study on carcinogenic potency of N-nitrosamines.

A mechanistic QSAR study on N-nitrosamines (NA) was performed with the aid of the semiempirical MINDO/3 method. Both the chemical reactivity and the transport in biological medium were taken into account. The parent NA molecules and their first reaction intermediates in the metabolic activation pathway were examined for possible determinants of the relative carcinogenic potency. The correlations found support the previous suggestions concerning the metabolic C alpha radical hydroxylation of NA. The role of transport properties in the early stage of NA biotransformation was also demonstrated.

Spectroscopic study of N-nitroso compounds decomposition in presence of DNA bases.

Decomposition kinetics of N-ethyl-N-nitrosourea (ENU) as well as that of N-methyl-N-nitroso-N'-nitroguanidine (MNNG) in water medium was studied by means of differential UV spectroscopy. Rate constants of the first order reaction were evaluated. The influence of DNA bases on decomposition rate of above-mentioned compounds was also estimated. With the help of NMR spectroscopy there were identified several products of decomposition as well as those of interaction of the studied compounds with DNA bases.