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.     

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.     

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      

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.     

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.     

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.     

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.     

A <Superscript>13</Superscript>C-detected <Superscript>15</Superscript>N double-quantum NMR experiment to probe arginine side-chain guanidinium <Superscript>15</Superscript>N<Superscript>η</Superscript> chemical shifts

Arginine side-chains are often key for enzyme catalysis, protein–ligand and protein–protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine ¹³C^ζ-detected NMR experiment in which a double-quantum coherence involving the two ¹⁵N^η nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two ¹⁵N^η; this new approach is shown to eliminate the previously deleterious line broadenings of ¹⁵N^η resonances caused by the partially restricted rotation about the C^ζ–N^ε bond. Consequently, sharp and well-resolved ¹⁵N^η resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine ¹⁵N^η nuclei becomes possible using the double-quantum experiment.     

<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.     

Rapid measurement of <Superscript>3</Superscript>J(H<Superscript>N</Superscript>–H<Superscript>α</Superscript>) and <Superscript>3</Superscript>J(N–H<Superscript>β</Superscript>) coupling constants in polypeptides

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.     

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.     

Synchronized reconstitution of muscle fibers,peripheral nerves and blood vessels by murine skeletal muscle-derived CD34<Superscript>−</Superscript>/45<Superscript>−</Superscript> cells

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34⁻/CD45⁻ (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2–8 × 10⁴); however, the number increased 10–20 fold (2–16 × 10⁵) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.     

Identification of CD133<Superscript>−</Superscript>/Telomerase<Superscript>low</Superscript> Progenitor Cells in Glioblastoma-Derived Cancer Stem Cell Lines

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. The CSC hypothesis implies that tumors are maintained by a rare subpopulation of CSC that gives rise to rapidly proliferating progenitor cells. Although the presence of progenitor cells is crucial for the CSC hypothesis, progenitor cells derived from GBM CSC are yet uncharacterized. We analyzed human CD133⁺ CSC lines that were directly derived from CD133⁺ primary astrocytic GBM. In these CSC lines, CD133⁺/telomerase^high CSC give rise to non-tumorigenic, CD133⁻/telomerase^low progenitor cells. The proliferation of the progenitor cell population results in significant telomere shortening as compared to the CD133⁺ compartment comprising CSC. The average difference in telomere length as determined by a modified multi-color flow fluorescent in situ hybridization was 320 bp corresponding to 4–8 cell divisions. Taken together, we demonstrate that CD133⁺ primary astrocytic GBM comprise proliferating, CD133⁻/telomerase^low progenitor cell population characterized by low telomerase activity and shortened telomeres as compared to CSC.     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

Sequence specific <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a cataract-related variant G57W of human γS-crystallin

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [¹⁵N–¹H]-HSQC spectrum recorded with uniformly ¹³C/¹⁵N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.