Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

Three's company: component structures bring a closer view of tripartite drug efflux pumps

Bacterial multidrug resistance is a serious clinical problem and is commonly conferred by tripartite efflux 'pumps' in the prokaryotic cell envelope. Crystal structures of the three components of a drug efflux pump have now been solved: the outer membrane TolC exit duct in the year 2000, the inner membrane AcrB antiporter in 2002 and the periplasmic adaptor MexA in 2004. These structures have enhanced our understanding of the principles underlying pump assembly and operation, and present pumps as new drug targets.     

Effects of frost hardening on the composition of galactolipids and phospholipids occurring during isolation of chloroplast thylakoids from needles of scots pine

Frost hardening of seedlings of Scots pine (Pinus sylvestris) at a non-freezing temperature of 4°C resulted in a 2-fold increase of the acyl lipids of the needles. This was because of increases in phospholipids and triglycerides. The galactolipid content of the needles was almost the same in unhardened and frost-hardened seedlings. In unhardened seedlings the mol ratio of monogalactosyl diacylglycerol (MGDG) to digalactosyl diacylglycerol (DGDG) was 1.7 ± 0.3 and 0.9 ± 0.2 in needles and isolated thylakoids, respectively. Corresponding ratios for frost-hardened seedlings were 1.5 ± 0.2 and 0.3 ± 0.03. The lower ratios found in isolated thylakoids, particularly in thylakoids from frost-hardened seedlings, are suggested to depend on the enzyme galactolipid: galactolipid galactosyltransferase being active during the isolation procedure. This is deduced from the result that the content of MGDG decreased and that of DGDG and 1.2 diglycerides increased. Needles of Scots pine also contain phospholipidase D. This enzyme was active during thylakoid preparation, particularly after frost hardening, as judged from the large amount of phosphatidic acid found the in thylakoid fraction isolated from frost-hardening needles. The fatty acid composition of the acyl lipids showed no major changes due to hardening at non-freezing temperature.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

Liquid chromatography tandem mass spectrometry method for the quantitation of NTBC (2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione) in plasma of tyrosinemia type 1 patients

In this study, we describe a bioanalytical method for quantification of NTBC in plasma of patients with hereditary tyrosinemia type 1 (HT-1) using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). After protein precipitation with acetonitrile including Mesotrione as internal standard, separation of NTBC was achieved by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. NTBC recovery in the developed method was found to be more than 90%. The lower limit of quantification was calculated to be 0.35 μM. The intra-day and inter-day precision of three different quality control samples (measured as RSD%) was less than 10% and 15%, respectively. The standard calibration curves showed good linearity within the range of 2.5–40 μM and the determined correlation coefficients were r² ≥ 0.995. This presented method is rapid, sensitive, specific and suitable for clinical practice and research.     

Antifungal effect of different methyl and propyl paraben mixtures on the treatment of paper biodeterioration

With many important artistic works and documents made of paper, and thus susceptible to biodeterioration by fungi, research is required in an effort to replace toxic chemical products with other more benign ones. In this work the antifungal effect of methyl and propyl paraben mixtures at different concentrations was evaluated. The fungi used in the experiments were a Cladosporium species and Penicillium corylophilum, both of which are well known as paper-biodeteriorating fungi. The results demonstrate that a mixture of 0.5% methyl paraben and 1% propyl paraben, in 85% ethanolic solution, is the lowest concentration necessary to provide an efficient antifungal action. A deacidification agent, 5% calcium propionate, was added to this mixture to produce a multi-purpose formulation to treat acidification and fungal contamination of paper documents. Tests carried out on paper samples before and after application of this mixture showed only a minor increase in yellowing and a slight decrease in tensile strength, while substantially raising the pH, and thus the alkaline reserve, and also a slight increase in the percentage of deformation.     

Thoracic aortic aneurysm patients with Chlamydophila pneumoniae infection showed a shift in trace element levels in serum and diseased aortic tissue

Few studies have been performed on trace elements in tissues and serum in cardiovascular disease and none in aortic aneurysm. In this study the concentrations of 10 trace elements were determined in serum and aneurysmatic aortic tissue from 23 patients undergoing thoracic surgery. Macroscopically, normal thoracic aortic tissue specimens from 10 forensic autopsies and serum from 23 healthy blood donors served as controls. DNA from the intracellular respiratory pathogen Chlamydophila pneumoniae (C. pneumoniae), which may be involved in the pathogenesis of atherosclerosis, was found in 26% (6/23) of the patients but in none of the controls. The serum copper/zinc ratio, a well-known marker of ongoing infection and/or inflammation, was higher (26%, p<0.001) in aneurysm patients. C. pneumoniae requires iron for its growth. In our aneurysm patients iron was higher in serum (by 54%, p<0.001) and aneurysmal tissue (by 60%, p<0.001). Although calcium was lower in patient sera (by 8%, p<0.001), it tended to be higher (by 20%, ns) in aneurysmatic tissue. In addition, mercury concentrations in serum and aneurysmatic tissue were positively correlated (r=0.51, p<0.05). Moreover, C. pneumoniae-positive aneurysmatic tissues had lower concentrations of manganese (46%, p<0.05) and zinc (26%, ns) but a higher concentration of mercury (50%, p<0.05) than C. pneumoniae-negative aneurysmatic tissues. In conclusion, aneurysm patients showed a shift in trace element levels in serum and in the diseased part of the aorta, the pattern being partly different in C. pneumoniae-positive compared with C. pneumoniae-negative patients. The results are compatible with active infection and/or inflammation, possibly initiated by C. pneumoniae.