Heightened endotoxin susceptibility of monocytes and neutrophils during familial Mediterranean fever

Familial Mediterranean fever (FMF) is a relapsing autoinflammatory disorder, caused by various mutations in the MEFV gene, which encodes a protein called pyrin, expressed in neutrophils and activated monocytes. Induction of monocyte endotoxin tolerance is observed in FMF patients during attack, whereas monocytes from patients in the attack-free period failed to induce lipopolysaccharide tolerance and exhibited heightened sensitivity to bacterial endotoxin. In this study, we demonstrated that impaired lipopolysaccharide tolerance induction in attack-free FMF patients correlates with both increased lipopolysaccharide-induced proinflammatory cytokine synthesis polarization and a different time-course pattern of lipopolysaccharide-induced changes on monocytic surface expression of CD14 and CD11b coreceptors. We found that this pattern is characterized either by delayed turnover of CD14 or increased surface retention of CD11b receptors on monocytes during stimulation with lipopolysaccharide. In addition, enhancement of lipopolysaccharide-induced apoptosis of neutrophils was observed in FMF patients, and was confirmed based on the fact that neutrophils from FMF patients previously unexposed to Salmonella enteritidis exhibited heightened susceptibility to the lipopolysaccharide of this pathogen similar to that of patients infected with this species.     

Short-range interactions and size of ligands bound to DNA strongly influence adsorptive phase transition caused by long-range interactions

Long-range interaction between all the ligands bound to DNA molecule may give rise to adsorption with the character of phase transition of the first kind (D. Y. Lando, V. B. Teif, J. Biomol. Struct & Dynam. 18, 903-911 (2000)). In this case, the binding curve, c(c(o)), is characterized by a sudden change of the relative concentration of bound ligands ((c)) at a critical concentration of free (unbound) ligands, c(o)=c(ocr), from a low c value to a high one where c(o) is molar concentration of free ligands. Such a transition might be caused by some types of DNA condensation or changes in DNA topology. For the study of the conditions necessary for adsorption with the character of phase transition, a calculation procedure based on the method of the free energy minimum is developed. The ligand size and two types of interactions between ligands adsorbed on DNA molecule are taken into consideration: long-range interaction between all the ligands bound to DNA and contact interactions between neighboring ligands. It was found that a) Stronger long-range interaction is required for longer ligands to induce phase transition that is occurred at greater c(ocr) values; b) Pure contact interaction between neighboring ligands can not itself initiate phase transition. However contact cooperativity strongly decreases the threshold value of energy of long-range interaction necessary to give rise to the transition.     

Low-Dose Electron-Beam Irradiation for the Improvement of Biofilm Formation by Probiotic Lactobacilli

The effects of 50–150 gray electron-beam irradiation on the biofilm-formation ability and cell surface hydrophobicity of the commercial strain, Lactobacillus acidophilus DDS®-1, from Lacto-G (a marketed synbiotic formulation) and the putative probiotic, L. rhamnosus Vahe, were evaluated. No significant changes in cell surface hydrophobicity were found after irradiation, while increases in biofilm-formation abilities were documented for both investigated microorganisms 0.22 ± 0.03 vs. 0.149 ± 0.02 (L. rhamnosus Vahe, 150 Gy) and 0.218 ± 0.021 vs. 0.17 ± 0.012 (L. acidophilus DDS®-1, 150 Gy). Given this, the use of electron-beam irradiation (50–100 Gy) for the treatment of L. rhamnosus Vahe and L. acidophilus DDS®-1 cells may be considered in product sterilization, quality improvement, and packaging practices.

     

Checklist of the freshwater fishes of Armenia,Azerbaijan and Georgia

We present a critical checklist of freshwater fish species found so far in the countries of Armenia, Azerbaijan and Georgia. In total 119 freshwater fishes are recorded. There are 40, 86 and 96 species currently known for Armenia, Azerbaijan and Georgia respectively. From these 119 species, seven are endemic and seven species are alien. From the alien species, only three (Carassius gibelio, Gambusia holbrooki and Pseudorasbora parva) can be considered as widespread and invasive. There are four species (Gasterosteus aculeatus, Gobio artvinicus, Perca fluviatilis and Salmo gegarkuni) that are translocated within the region. Seven species are confirmed or recorded for the first time including G. artvinicus and Oxynoemacheilus veyselorum for Armenia, Azerbaijan and Georgia, Capoeta kaput and Rhinogobius lindbergi for Azerbaijan and Georgia, Capoeta razii for Azebaijan, Oxynoemacheilus cemali and Squalius agdamicus for Georgia. In this checklist, Acipenser colchicus is treated as a synonym of Acipenser persicus. Sand smelts of the Black and Caspian Sea basin are identified as Atherina caspia and Clupeonella caspia is treated as a synonym of Clupeonella cultriventris. Coregonus sevanicus is listed as Coregonus sp. until the situation of Sevan whitefish is better understood. Capoeta sevangi and Capoeta ekmekciae are synonyms of Capoeta capoeta. The fish often identified as Capoeta capoeta gracilis from rivers south of the Kura most likely belong to C. razii. The Black and Caspian Sea Rutilus populations are treated as conspecific, therefore R. kutum is a junior synonym of R. frisii. Oxynoemacheilus veyseli is valid as O. veyselorum. We list the alien Rhinogobius species as R. lindbergi, however the name is provisional and needs further confirmation. All Squalius species from the Kura River drainage are identified as S. agdamicus, however in the Aras, it is replaced by S. turcicus. Squalius orientalis is treated as a valid species restricted to the eastern Black Sea basin. The four forms of Lake Sevan trout (Salmo ischchan, S. gegarkuni, S. danilewskii and S. aestivalis) are treated as valid species, two of them (S. ischchan and S. danilewskii) are extinct. Rutilus sojuchbulagi from Azerbaijan is also extinct.     

Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.     

Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays

The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on solid supports.     

Compensation of DNA stabilization and destabilization effects caused by cisplatin is partially disturbed in alkaline medium

Binding of the antitumor compound cisplatin to DNA locally distorts the double helix. These distortions correlate with a decrease in DNA melting temperature (Tm). However, the influence of cisplatin on DNA stability is more complex because it decreases the DNA charge density. In this way, cisplatin increases the melting temperature and partially compensates for the destabilizing influence of structural distortions. The stabilization is stronger at low Na+ ion concentration. Due to this compensation, the total decrease in the DNA melting temperature after cisplatin binding is much lower than the decrease caused by the distortions themselves, especially at low [Na+]. It is shown in this study that, besides Na+ concentration, pH also strongly influences the value of a change in the melting temperature caused by cisplatin. In alkaline medium (pH=10.5-10.8), a fall in the melting temperature caused by platination is enhanced several times with respect to neutral medium. Such a stronger drop in Tm is explained by a decrease in pK values of base pairs caused by lowering the charge density under platination that facilitates proton release. At neutral pH, the proton release is low for both control and platinated DNA and does not influence the melting behavior. Therefore, lowering in the charge density under platination, besides stabilization, gives additional destabilization just in alkaline medium. Destabilization caused by structural distortions due to this pH induced compensation of stabilizing effect is more pronounced. In the presence of carbonate ion, destabilization caused by high pH value is strengthened. As a decrease in DNA charge density, interstrand crosslinking caused by cisplatin also increases the DNA stability due to loss in the entropy of the melted state. However, computer modeling of DNA stability demonstrates that interstrand crosslinks formed by cisplatin do not stabilize long DNA. It is shown that the increase in Tm caused by interstrand crosslinking itself is compensated for by a local destabilization of the double helix at the sites of location of interstrand crosslinks formed by cisplatin.     

The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle

Effects of the cytoplasmic tail (CT) of human immunodeficiency virus type 1 Env on the process of membrane fusion were investigated. Full-length Env (wild type [WT]) and Env with its CT truncated (DeltaCT) were expressed on cell surfaces, these cells were fused to target cells, and the inhibition of fusion by peptides that prevent Env from folding into a six-helix bundle conformation was measured. For both X4-tropic and R5-tropic Env proteins, DeltaCT induced faster fusion kinetics than did the WT, and peptides were less effective at inhibiting DeltaCT-induced fusion. We tested the hypothesis that the inhibitory peptides were less effective at inhibiting DeltaCT-induced fusion because DeltaCT folds more quickly into a six-helix bundle. Early and late intermediates of WT- and DeltaCT-induced fusion were captured, and the ability of peptides to block fusion when added at the intermediate stages was quantified. When added at the early intermediate, the peptides were still less effective at inhibiting DeltaCT-induced fusion but they were equally effective at preventing WT- and DeltaCT-induced fusion when added at the late intermediate. We conclude that for both X4-tropic and R5-tropic Env proteins, the CT facilitates conformational changes that allow the trimeric coiled coil of prebundles to become optimally exposed. But once Env does favorably expose its coiled coil to inhibitory peptides, the CT hinders subsequent folding into a six-helix bundle. Because of this facilitation of maximal exposure and hindrance of bundle formation, the coiled coil is optimally exposed for a longer time for WT than for DeltaCT. This accounts for the greater peptide inhibition of WT-induced fusion.     

Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion

Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37 degrees C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23 degrees C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37 degrees C from the 23 degrees C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23 degrees C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23 degrees C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23 degrees C was prolonged, more cells quickly fused upon the raising of the temperature to 37 degrees C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form.     

Hypothalamic Proline-Rich Polypeptide is an Oxidative Burst Regulator

The AGAPEPAEPAQPGVY proline-rich polypeptide (PRP) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. PRP possesses immune-modulating activity, preventing the death of Gram-negative bacteria-infected mice. Here we show that PRP does not affect human peripheral blood neutrophlis and monocytes phagocytosis but dramatically enhances spontaneous or fMLP- and PMA-induced, and also phagocytosis-dependent, oxidative burst. We demonstrated the regulatory role of PRP on the oxidative burst induction of normal and relapsing inflammatory disease (Behcets disease and familial Mediterranean fever) neutrophils and monocytes. Our results suggest a previously undescribed role for the hypothalamic peptide within primary activated neutrophils and monocytes, since we provide evidence that PRP can differentially regulate both chemotaxis- and phagocytosis-dependent oxidative burst in normal and inflammatory disease effector cells.