CD14<Superscript>++</Superscript>CD16<Superscript>−</Superscript> and CD14<Superscript>+</Superscript>CD16<Superscript>+</Superscript> human monocyte adhesion to endothelial cells

Based on the difference in the CD14 and CD16 expression, two subsets of monocytes were identified in human and other mammalian blood. These subsets have different patterns of adhesion molecules and chemokine receptors that suggests the different mode of their interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14⁺CD16⁺ and CD14⁺⁺CD16⁻ monocytes to adhere to endothelial cell monolayer in presence or absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14⁺CD16⁺ monocytes had a higher level of adhesion to intact monolayer of endothelial cells than CD14⁺⁺CD16⁻ monocytes. Adhesion of CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes significantly increased in the presence of TNFα or its combination with other cytokines. IFNγ and IL-4 alone did not affect the adhesion of monocytes. These results show that CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes can be recruited to the inflamed endothelium, but CD14⁺CD16⁺ monocytes adhere to endothelial cells without inflammations twice as strongly as CD14⁺⁺CD16⁻ monocytes.     

Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2      

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase β1 subunit interacts with M2 proteins of influenza A and B viruses and affects the virus replication

Interplay between the host and influenza virus has a pivotal role for the outcome of infection. The matrix proteins M2/BM2 from influenza (A and B) viruses are small type III integral membrane proteins with a single transmembrane domain, a short amino-terminal ectodomain and a long carboxy-terminal cytoplasmic domain. They function as proton channels, mainly forming a membrane-spanning pore through the transmembrane domain tetramer, and are essential for virus assembly and release of the viral genetic materials in the endosomal fusion process. However, little is known about the host factors which interact with M2/BM2 proteins and the functions of the long cytoplasmic domain are currently unknown. Starting with yeast two-hybrid screening and applying a series of experiments we identified that the β1 subunit of the host Na⁺/K⁺-ATPase β1 subunit (ATP1B1) interacts with the cytoplasmic domain of both the M2 and BM2 proteins. A stable ATP1B1 knockdown MDCK cell line was established and we showed that the ATP1B1 knockdown suppressed influenza virus A/WSN/33 replication, implying that the interaction is crucial for influenza virus replication in the host cell. We propose that influenza virus M2/BM2 cytoplasmic domain has an important role in the virus-host interplay and facilitates virus replication.     

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

Molecular modeling of the rabbit colonic (HKα2a) H<Superscript>+</Superscript>, K<Superscript>+</Superscript> ATPase

A model of the HK2a subunit of the rabbit colonic H⁺, K⁺ ATPase has been generated using the crystal structure of the Ca⁺² ATPase as a template. A pairwise sequence alignment of the deduced primary sequences of the two proteins demonstrated that they share 29% amino acid sequence identity and 47% similarity. Using O (version 7) the model of HK2a was constructed by interactively mutating, deleting, and inserting the amino acids that differed between the pairwise sequence alignment of the Ca⁺² ATPase and HK2a. Insertions and deletions in the HK2a sequence occur in apparent extra-membraneous loop regions. The HK2a model was energy minimized and globally refined to a level comparable to that of the Ca⁺² ATPase structure using CNS. The charge distribution over the surface of HK2a was evaluated in GRASP and possible secondary structure elements of HK2a were visualized in BOBSCRIPT. Conservation and placement of residues that may be involved in ouabain binding by the H⁺, K⁺ ATPase were considered and a putative location for the subunit was postulated within the structure.Figure Possible architecture of the HK2a subunit. The residue in green is the lysine (position 517, Fig. 1) that lies in the nucleotide binding pocket and the residue in red is the aspartic acid at the phosphorylation site (position 385). Based on an alignment with the Ca⁺² ATPase, ten transmembrane helices were modeled into HK2a. The ten transmembrane helices are drawn as rods and shown in different colors for clarity. From left to right, the transmembrane helix designations are M10 (blue), M7 (gray), M8 (purple), M9 (orange), M5 (pink), M6 (green), M3 (brown), M4 (cyan), M2 (teal), and M1 (almond).     

Transmembrane transport of K<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> during pollen grain activation in vivo and in vitro

We studied the possibility of K⁺ and Cl⁻ efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl⁻ efflux; potassium channel inhibitor TEA (tetraethylammonium chloride) partially reduced K⁺ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba²⁺ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K⁺ and Cl⁻ release. An important role in these processes is played by NPPB-, TEA- and Ba²⁺-sensitive plasmalemma ion channels.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Synthesis of the β-amyloid fragment <Superscript>5</Superscript>RHDSGY<Superscript>10</Superscript> and its isomers

The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGY peptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(β-Asp)SGY (or RHDSGY during synthesis of RH(β-Asp)SGY) and RH(Asp-imide) SGY was solved via selection of individual reagents for removal of Fmoc groups from α-amino groups of the growing peptide chain.     

Dissociation of Lipid Components and Reconstitution at – 75° C of Mg<Superscript>2+</Superscript> Dependent,Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> Stimulated,Adenosine Triphosphatase in Rat Brain

MEMBRANE enzymes, because of their lipid content, are insoluble in water and usually solubilized as micelles in aqueous solution by detergents for biochemical study. Direct study of lipid components by means of organic liquids in which they dissolve leads at once to the denaturation of the enzyme. At temperatures appreciably colder than 0° C, however, organic solvents may leave enzymatic activity intact^1–3, making it possible to study enzyme reactions.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

Role of unusual CD4<Superscript>+</Superscript> CD28<Superscript>−</Superscript> T cells in acute coronary syndrome

Acute coronary syndrome (ACS) is a group of clinical symptoms that results from complete or partial occlusive thrombus, which is caused by coronary an atherosclerotic plaque rupture or erosion. According to a recent study, CD4⁺ CD28⁻ T cells are found in atherosclerotic plaques and the peripheral circulation blood in patients with ACS, these cells play an important role in plaque ruptures. CD4⁺ CD28⁻ T cells are an unusual subset of helper cells, which expand and have harmful effects in ACS. In this review, we discuss the current issues on the generation of CD4⁺ CD28⁻ T cells and focus on their phenotypic and functional characteristics relevant to the development of cardiovascular events. Targeting the CD4⁺ CD28⁻ T cells subset in ACS could provide novel therapeutic means to prevent acute life-threatening coronary events.     

CD45<Superscript>−</Superscript>CD14<Superscript>+</Superscript>CD34<Superscript>+</Superscript> murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term <Emphasis Type="Italic">in vitro</Emphasis> culture

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45⁻CD14⁺CD34⁺ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45⁻CD14⁺ CD34⁺ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.     

Anionic Selectivity Sequence of the Cl<Superscript>−</Superscript>–H<Superscript>+</Superscript> Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na⁺/H⁺ exchanger has been the only unequivocally demonstrated H⁺-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl⁻–H⁺ symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl⁻–H⁺ symporter is NO₃⁻ > Br⁻ > Cl⁻ >> I⁻ = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 μM inhibits the gluconate-dependent alkalinization by 30 ± 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization. Special issue article in honor of Dr. Ricardo Tapia.