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.     

APRIL depletion induces cell cycle arrest and apoptosis through blocking TGF-β1/ERK signaling pathway in human colorectal cancer cells

It is well documented that a proliferation-inducing ligand (APRIL), a newly found member of tumor necrosis factor superfamily, overexpressed in the majority of malignancies, plays a potential role in the occurrence and development of these tumors. Herein, we demonstrated that APRIL depletion by using RNA interference in human colorectal cancer (CRC) COLO 205 and SW480 cells resulted in cell proliferation inhibition and evoked cell cycle arrest in G0/G1 phase and apoptosis, coupled with decrease in CDK2, Cyclin D1, Bcl-2 expression and an increase of p21 and Bax expression. In addition, the decreased expression of transforming growth factor-β1 (TGF-β1) and p-ERK was also showed in siRNA-APRIL transfected COLO 205 and SW480 cells, whereas the protein expression levels of Smad2/3, p-Smad2/3, and ERK were not significantly changed. Taken together, our results indicate that APRIL depletion induces cell cycle arrest and apoptosis partly through blocking noncanonical TGF-β1/ERK, rather than canonical TGF-β1/Smad2/3, signaling pathway in CRC cells. Moreover, our study highlights APRIL as a potential molecular target for the therapy of CRC.     

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.     

Defective CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis,counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4⁺CD25⁺ T_reg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T_reg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4⁺CD25⁺ T cells in the central and peripheral lymphoid tissues. In addition, CD4⁺CD25⁺ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4⁺CD25⁺ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T_reg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T_reg cells and accessory cells. Our results demonstrate that the decrease in T_reg cell activity in CIA is counter-regulated by endogenous IFN-γ.     

γ-Tocotrienol induces growth arrest through a novel pathway with TGFβ2 in prostate cancer

Regions along the Mediterranean and in southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of tocotrienols. Here we examine the potential of γ-tocotrienol (GT3) to reduce prostate cancer proliferation and focus on elucidating pathways by which GT3 could exert a growth-inhibitory effect on prostate cancer cells. We find that the γ and δ isoforms of tocotrienol are more effective at inhibiting the growth of prostate cancer cell lines (PC-3 and LNCaP) compared with the γ and δ forms of tocopherol. Knockout of PPAR-γ and GT3 treatment show inhibition of prostate cancer cell growth, through a partially PPAR-γ-dependent mechanism. GT3 treatment increases the levels of the 15-lipoxygenase-2 enzyme, which is responsible for the conversion of arachidonic acid to the PPAR-γ-activating ligand 15-S-hydroxyeicosatrienoic acid. In addition, the latent precursor and the mature forms of TGFβ2 are down-regulated after treatment with GT3, with concomitant disruptions in TGFβ receptor I, SMAD-2, p38, and NF-κB signaling.     

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.     

IL-4 inducibility in NKT cells,naïve CD4<Superscript>+</Superscript> T cells and TCR-γ δ T cells

NKT cells, na?ve CD4(+) T cells, and TCR-gammadelta T cells belong to distinct T cell lineages but all express T cell receptors generated through random combinatorial joining of V-(D)-J genes. These distinct lineage T cells also possess the property of promptly activating the IL-4 gene upon T cell receptor stimulation. A comparative accounting of features as they pertain to IL-4 inducibility in these three distinct lineage T cells is provided here.     

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.     

HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway

Previous studies have revealed the elevated serum levels of High-mobility group box-1(HMGB1) and the interferon-γ (IFN-γ)-induced proliferation of renal mesangial cells in patients or experimental animals with systemic lupus erythematosus (SLE). However, it is still not elucidated whether HMGB1 involves in the pathogenesis of lupus nephritis (LN) and mediates IFN-γ-induced mesangial cell proliferation. Therefore, in the present study we demonstrated HMGB1 mRNA and protein levels were increased in the glomeruli of LN patients and BXSB mice. HMGB1 increased the proliferation index of mouse mesangial cells (MMC) that was accompanied with the up-regulation of cyclin D1, CDK4 and the down-regulation of p16, subsequently promoting the transition from the G0/G1 to S stage. Inhibition of HMGB1 by a specific short hairpin RNA vector prevented cyclin D1/CDK4/p16 up-regulation and attenuated IFN-γ-induced MMC cell proliferation and PCNA (proliferating cell nuclear antigen, PCNA) expression. These findings indicate that HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway and promoting the cell cycle transition from G1 to S stage.     

NO<Subscript>3</Subscript><Superscript>−</Superscript>-Driven SO<Subscript>4</Subscript><Superscript>2−</Superscript> Production in Freshwater Ecosystems: Implications for N and S Cycling

Massive anthropogenic acceleration of the global nitrogen (N) cycle has stimulated interest in understanding the fate of excess N loading to aquatic ecosystems. Nitrate (NO₃ ⁻) is traditionally thought to be removed mainly by microbial respiratory denitrification coupled to carbon (C) oxidation, or through biomass assimilation. Alternatively, chemolithoautotrophic bacterial metabolism may remove NO₃ ⁻ by coupling its reduction with the oxidation of sulfide to sulfate (SO₄ ²⁻). The NO₃ ⁻ may be reduced to N₂ or to NH₄ ⁺, a form of dissimilatory nitrate reduction to ammonium (DNRA). The objectives of this study were to investigate the importance of S oxidation as a NO₃ ⁻ removal process across diverse freshwater streams, lakes, and wetlands in southwestern Michigan (USA). Simultaneous NO₃ ⁻ removal and SO₄ ²⁻ production were observed in situ using modified “push-pull” methods in nine streams, nine wetlands, and three lakes. The measured SO₄ ²⁻ production can account for a significant fraction (25–40%) of the overall NO₃ ⁻ removal. Addition of ¹⁵NO₃ ⁻ and measurement of ¹⁵NH₄ ⁺ production using the push–pull method revealed that DNRA was a potentially important process of NO₃ ⁻ removal, particularly in wetland sediments. Enrichment cultures suggest that Thiomicrospira denitrificans may be one of the organisms responsible for this metabolism. These results indicate that NO₃ ⁻-driven SO₄ ²⁻ production could be widespread and biogeochemically important in freshwater sediments. Removal of NO₃ ⁻ by DNRA may not ameliorate problems such as eutrophication because the N remains bio-available. Additionally, if sulfur (S) pollution enhances NO₃ ⁻ removal in freshwaters, then controls on N processing in landscapes subject to S and N pollution are more complex than previously appreciated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detailed cell cycle analysis in human lymphocytes; Application to γ-irradiated cells

Summary A method based on BrdU incorporation for analyzing in detail the kinetics of the cell cycle is described. The S phase has been subdivided into five subphases, each recognizable by their BrdU incorporation pattern at metaphase. The method can be useful for the study of abnormal cell cycles, and may have particular application in mutagenesis studies concerning the various subphases of the S phase, without using synchronization techniques. An application of the method is described, showing that -irradiation, during the course of the S phase, leads to a lack of cells which were in early S phase at the time of irradiation. This finding can be related either to a higher lethality at this stage of the cell cycle or to a delay in completion of DNA replication after irradiation.Hoider of a C.E.C. scholarship     

A simple strategy for <Superscript>13</Superscript>C,<Superscript>1</Superscript>H labeling at the Ile-γ2 methyl position in highly deuterated proteins

A straightforward approach for the production of highly deuterated proteins labeled with ¹³C and ¹H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile ¹³C^γ2,¹H^γ2 HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile ¹³C^δ1,¹H^δ1 spectra. This labeling scheme adds to a growing list of different approaches that are significantly impacting the utility of solution NMR spectroscopy in studies of supra-molecular systems.     

α-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant O<Subscript>2</Subscript><Superscript>−.</Superscript>-generation

The antioxidant -lipoic acid (ALA) has been shown to affect a variety of biological processes associated with oxidative stress including cancer. We determined in HT-29 human colon cancer cells whether ALA is able to affect apoptosis, as an important parameter disregulated in tumour development. Exposure of cells to ALA or its reduced form dihydrolipoic acid (DHLA) for 24 h dose dependently increased caspase-3-like activity and was associated with DNA-fragmentation. DHLA but not ALA was able to scavenge cytosolic O₂^–. in HT-29 cells whereas both compounds increased O₂^{– .}-generation inside mitochondria. Increased mitochondrial O₂^{– .}-production was preceded by an increased influx of lactate or pyruvate into mitochondria and resulted in the down-regulation of the anti-apoptotic protein bcl-X_L. Mitochondrial O₂^–.-generation and apoptosis induced by ALA and DHLA could be prevented by the O₂^{– .}-scavenger benzoquinone. Moreover, when the lactate/pyruvate transporter was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoate, ALA- and DHLA-induced mitochondrial ROS-production and apoptosis were blocked. In contrast to HT-29 cells, no apoptosis was observed in non-transformed human colonocytes in response to ALA or DHLA addition. In conclusion, our study provides evidence that ALA and DHLA can effectively induce apoptosis in human colon cancer cells by a prooxidant mechanism that is initiated by an increased uptake of oxidizable substrates into mitochondria.