Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the “active management” of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.     

A relationship between RP4 plasmid acquisition and phenotypic changes in Pseudomonas fluorescens R2fN

The physiological behaviour of Pseudomonas fluorescens strain R2fN was compared to that of transconjugants [R2fN(RP4)], and two aggregation phenotypes were identified (Agr^– and Agr⁺). Agr⁺ phenotype is characterized by the appearance of macroscopic aggregates when cells are growing in liquid media. Transconjugants exhibited Agr⁺ phenotype whereas wild type strain represented Agr^–. Evidence is presented to support correlation between Agr⁺ phenotype acquisition and the presence of the broad-host range plasmid RP4 in strain R2fN. In addition, according to bacterial adherence to hydrocarbon test the transconjugant cells appeared to be very hydrophilic whereas wild type R2fN cells were hydrophobic.     

Secondary screening of transconjugant clones mediated by a lytic bacteriophage

D.D.G.M. and A.L.Z each contributed to 50%.Due to the lack of suitable chromosomal markers, antibiotics could not be used to select transconjugant clones arising from matings between two Pseudomonas strains. As an alternative, a method based on the specificity of a lytic bacteriophage for the recipient strain was used successfully. © Rapid Science Ltd. 1998     

Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Protein Profiling of the Medicinal Leech Salivary Gland Secretion by Proteomic Analytical Methods

Protein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One-dimensional (1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to 483 kD. 2D-electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values. SELDI-mass spectrometry analysis using the ProteinChip. System based on chromatography surfaces of strong anion or weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of SELDI-MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active components of leech saliva secretion.     

Affinity-based proteomic profiling: problems and achievements

Affinity chromatography becomes a more and more popular method used in proteomic studies for separation of various groups of proteins (subproteomes). The review highlights the role of affinity chromatography fractionation for proteomic profiling of the most of intensively studied groups of proteins including cyclic nucleotide-binding proteins, protein kinases (kinomes), phosphoproteins, glycoproteins, ubiquitinated proteins. Special attention is paid to the use of affinity chromatography for the characterization of small-molecule protein targets. The latter is especially important for the elucidation of direct protein targets of potential drug substances for evaluation of their possible side-effects or additional pharmacological application.     

Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system

The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.     

Proteome of the human HaCaT keratinocytes: Identification of the oxidative stress proteins after sodium dodecyl sulpfate exposur

Oxidative stress is a universal response of the skin cell damage of various origins. Sodium dodecyl sulfate (SDS, sodium lauryl sulfate) is an anionic surfactant commonly used as an emulsifying detergent in household cleaners. Sodium dodecyl sulfate is the reference compound for testing toxicity on cellular skin models. The effect of sodium dodecyl sulfate in sub toxic dose 25 μg/mL during 48 h on the protein profile of human keratinocytes HaCaT was studied by tandem mass spectrometry with electrospray ionization. In total, 1064 proteins were found in immortalized human keratinocytes HaCaT, of which about 80% were identified by two or more peptides. The change of the 217 proteins content was revealed, among them 39 according to Gene Ontology are associated with oxidative stress. It has been found that sodium dodecyl sulfate leads to a decrease in the number of proteins/peptides containing carboxymethylated and/or carboxyethylated lysine. We concluded about the promising of the cells redox-balance analysis at testing chemicals in the doses, which do not lead to a decrease in their viability. Possible involvement of sodium dodecyl sulfate in the development of cutaneous neoplasia is discussed.