The redox state of glutathione, cysteine and homocysteine in the extracellular fluid in the skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

Enantiomeric separations of amino alcohols by packed-column SFC on Hypercarb with L-(+)-tartaric acid as chiral selector

The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min.     

Relative contribution of residential and occupational magnetic field exposure over twenty-four hours among people living close to and far from a power line

This study sought to estimate the relative contribution of exposure to 50 Hz magnetic fields experienced at home, at work/school, or elsewhere to the total exposure over 24 hr. Personal exposure meters were carried by 97 adults and children in the Stockholm area. About half of the subjects lived close (<50 m) to a transmission line and half far (>100 m) away. Spot measurements and calculations for the residential exposure were also made. For subjects living<50 m from the line, the exposure at home contributed about 80% of the total magnetic field exposure, measured in mT-hours. Adults living far away experienced only 38% of the total exposure at home, but children still received 55%. Subjects with low time-weighted average (TWA) exposure both at home and at work spent 84% of their time in fields <0.1 microT, and those with high TWA at both locations spent 69% of their time in fields > or = 0.2 microT. This contrast was diluted if only exposure at one location was considered. For spot measurements and calculations of the residential exposure, both sensitivity and specificity was good. However, the intermediate field exposure category (0.1-0.19 microT) showed poor correlation to the 24 hr personal measurements.     

Folding of a small helical protein using hydrogen bonds and hydrophobicity forces

A reduced protein model with five to six atoms per amino acid and five amino acid types is developed and tested on a three-helix-bundle protein, a 46-amino acid fragment from staphylococcal protein A. The model does not rely on the widely used Go approximation, which ignores non-native interactions. We find that the collapse transition is considerably more abrupt for the protein A sequence than for random sequences with the same composition. The chain collapse is found to be at least as fast as helix formation. Energy minimization restricted to the thermodynamically favored topology gives a structure that has a root-mean-square deviation of 1.8 A from the native structure. The sequence-dependent part of our potential is pairwise additive. Our calculations suggest that fine-tuning this potential by parameter optimization is of limited use.     

The role of the cell cycle on the efficiency of photochemical gene transfection

The efficiency of gene transfection mediated by nonviral vectors is limited because of nonoptimal intracellular trafficking of transfecting DNA. Most nonviral vectors deliver transfecting DNA into a cell through endocytosis. However, poor escape from endocytic vesicles and inefficient transport of DNA into the nucleus often limits a success of gene transfection. Photochemical transfection is a new method, based on light-induced permeabilisation of endocytic vesicles, liberating transfecting DNA into the cytosol, concurrently increasing the chances for DNA to enter the nucleus.The aim of this study was to investigate the role of the cell cycle for the efficiency of photochemical transfection. It was demonstrated that in asynchronous human colon carcinoma HCT 116 cells photochemical treatment increased the transfection mediated by the nonviral vectors, the cationic polypeptide polylysine and the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (beta AE-DMRIE/DOPE), by 30- and 2.5-fold, respectively. In aphidicolin-synchronised cells, photochemical transfection mediated by polylysine was dependent on the cell cycle: transfection level was 4-fold higher when illumination, inducing photochemical reactions, was performed during the G2/M phase as compared to the G1/early-S phase. The cell cycle influence on photochemical transfection mediated by beta AE-DMRIE/DOPE was very low: only 20% difference between G2/M and the G1/S phase was observed. We suggest that transgenes, photochemically liberated close/during mitosis, perhaps have the highest opportunity to enter the nucleus and be expressed. However, the dependence of photochemical transfection on the cell cycle might be partially disguised by various factors induced by photochemical treatment.     

Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7

Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.     

Virulence determinants and protective antigens of Francisella tularensis

Very little is known about virulence mechanisms of the highly virulent bacterium Francisella tularensis. Specific genetic features of F. tularensis have been obstacles for the development of effective tools for genetic manipulation. However, recent genomic sequencing and large-scale proteomic work have resulted in a substantial increase in the knowledge of F. tularensis. There is also a paucity of information on potential vaccine candidates. Recent work assessing the protective efficacy of the F. tularensis lipopolysaccharide has resulted in important contributions to the understanding of host-protective mechanisms. T-cell-mediated immunity appears to be crucial to protect against virulent F. tularensis strains. Few other vaccine candidates have been identified.     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

Genetic polymorphism and sequence evolution of an alternatively spliced exon of the glial fibrillary acidic protein gene,GFAP

Isoform GFAPepsilon of the human cytoskeletal protein GFAP carries, as the result of alternative splicing of exon 7a of GFAP, a novel 42-amino-acid-long C-terminal region with binding capacity for the presenilin proteins. Here we show that exon 7a is present in a variety of mammals but absent from GFAP of chicken and fish. Comparison of the mouse and human GFAP exons showed an increased rate of nonsynonymous nucleotide substitutions in exon 7a compared to the other exons. This resulted in 10 nonconservative and 2 conservative amino acid substitutions and suggests that exon 7a has evolved under different functional constraints. Exons 7a of humans and higher primates are 100% identical apart from alanine codon 426, which is conserved in only 9% of the human alleles, while 21 and 70% of the alleles, respectively, have a valine or a threonine codon at that position. Threonine represents a potential phosphorylation site, and positive selection of that effect could explain the high allele frequency.     

Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure

Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We found protein expression in the double deletion strain to be highly similar to wild-type. In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa). Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein. Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues. We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases. Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points. However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared. In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini. Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini.