Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time‐dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 µM . Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time‐dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration‐dependent, which correlates to the concentration‐dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration‐dependent killing is driven by the extent of the immediate permeabilizing action of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l⁻¹). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l⁻¹. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn²⁺) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.     

Determination of 8-iso-prostaglandin F(2alpha) in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.     

Comparison of element levels in minimal and complex yeast media

The cellular functions are strongly influenced by the composition of the environment. In particular, phenotypes of microbial strains are modulated by concentrations of ions in the culture medium, and differences in element levels may be responsible for a phenotypic variability observed when microbial strains are grown on synthetic versus complex media. In this report, we analyzed the levels of nine elements (magnesium, potassium, sodium, calcium, iron, copper, manganese, zinc, and phosphorus) and sulphate ions in commercially available peptone and yeast extract and compared them with those in yeast nitrogen base routinely used for preparation of synthetic minimal media. We observed that whereas some elements are present at similar levels, the levels of others differ by a factor as high as 20. The observed differences should be taken into account when interpreting different phenotypes observed for microbial strains grown on synthetic versus complex media.     

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.     

Carboxylate and aromatic active-site residues are determinants of high-affinity binding of ω-aminoaldehydes to plant aminoaldehyde dehydrogenases

The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet?al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.     

Control of spindle form and function in grasshopper spermatocytes

The influence of the mitotic organizing centers, the kinetochores and the polar organizers, in controlling the dynamic spindle form and function has been investigated in the primary spermatocytes of two grasshoppers, Arphia xanthoptera and Melanoplus differentialis. A new measure of the total birefringent material in the spindle is introduced—volume-birefringence. This measure avoids many of the problems associated with the traditional retardation measurements of spindle organization.—The number of chromosomes (and their kinetochores) in a spindle can be altered with a piezoelectric micromanipulator in three ways: 1) chromosomes can be removed permanently from the cell, 2) chromosomes can be detached from the spindle and allowed to reenter the spindle at a later time, and 3) chromosomes can be transferred from one spindle to another in cells containing two spindles. Such operations show the volume-birefringence of the spindle is proportional to the number of chromosomes in the spindle. A residual volume-birefringence is seen and attributed to the contribution of the polar organizers to spindle structure. The relative polar contribution differs in the two species. Chromosome motion and spindle elongation in anaphase are unaffected by the number of chromosomes in the spindle. The proportion of volume-birefringence associated with a kinetochore is used to estimate the number of microtubules one might expect to see if the birefringence of the spindle is of microtubular origin. These calculations predict about twice the number of microtubules per kinetochore than seen with the electron microscope. Reasons are suggested to explain this discrepancy.— It is argued that chromosome detachment releases spindle component subunits into the total subunit pool, but that these excess subunits do not influence the metaphase form nor the anaphase function of the spindle; therefore, spindle dynamics are under the direct control of the kinetochores and the polar organizing centers.     

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Background

Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3.

Results

Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98.

Conclusions

The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.

     

Identification and molecular characterization of <Emphasis Type="Italic">Theileria</Emphasis> sp. infecting red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) in northwestern Poland

Piroplasms from Theileria genus were detected in blood and spleen of red deer Cervus elaphus culled during the months of September 2004–January 2005 in northwestern Poland. The polymerase chain reaction revealed the presence of Theileria deoxyribonucleic acid in 88% (36 of 41) of the animals examined. Molecular characterization of the parasites based on large piece of 18S ribosomal ribonucleic acid gene containing hypervariable region V4 showed 99.9% similarity to two Theileria spp. sequences: Theileria sp. 3185/02 and Theileria capreoli BAB1158. Phylogenetic analysis confirmed that the three isolates cluster together with high bootstrap support. It is supposed that those pathogens can be classified as one group characteristic for the Eurasian continent, contrary to protozoon of Theileria from the T. cervi group, which are often found on the North American continent and can also infect the representatives of Cervidae. In conclusion, this study suggested that free-living C. elaphus in northwestern Poland are a competent reservoir of Theileria sp. ZS T04 C.e. parasites, although the vector of the piroplasms is still unknown.