Lysoplasmenylcholine increases neutrophil adherence to human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in release of choline lysophospholipids [lysophosphatidylcholine (lysoPtdCho) and lysoplasmenylcholine (lysoPlsCho)]. These amphiphilic metabolites have been implicated in arrhythmogenesis following the onset of myocardial ischemia, but studies examining their direct effects on the vasculature remain limited. We and others have shown that thrombin and lysoPtdCho can increase cell surface adhesion molecules and adherence of circulating inflammatory cells to the endothelium. This study supports our hypothesis that these changes may be mediated, at least in part, by lysoPlsCho, thus implicating this metabolite as an inflammatory mediator in the coronary vasculature and a modulator of the progression of atherosclerosis. Apical stimulation of HCAEC with thrombin resulted in the production and release of choline lysophospholipids from the apical surface of the HCAEC monolayer. Basolateral stimulation had no effect on choline lysophospholipid production or release from either the apical or basolateral surface of the HCAEC monolayer. Incubation of HCAEC with lysoPlsCho or lysoPtdCho resulted in similar increases in HCAEC surface expression of P-selectin and E-selectin. Furthermore, lysoPlsCho increased cell surface expression of P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with a time course similar to that of thrombin stimulation. Increased presence of cell surface adhesion molecules may contribute to the significant increase in adherence of neutrophils to either thrombin- or lysoPlsCho-stimulated HCAEC. These results demonstrate that the presence of thrombin at sites of vascular injury in the coronary circulation, resulting in increased choline lysophospholipid release from the HCAEC apical surface, has the potential to propagate vascular inflammation by upregulation of adhesion molecules and recruitment of circulating inflammatory cells to the endothelium. endothelium; arrhythmogenesis; inflammation; lysophospholipids     

Secretogranin III directs secretory vesicle biogenesis in mast cells in a manner dependent upon interaction with chromogranin A

Mast cells are granular immunocytes that reside in the body's barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the proteins that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) protein is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1-210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1.     

Proteomic Analysis of Microtubule-associated Proteins during Macrophage Activation

Classical activation of macrophages induces a wide range of signaling and vesicle trafficking events to produce a more aggressive cellular phenotype. The microtubule (MT) cytoskeleton is crucial for the regulation of immune responses. In the current study, we used a large scale proteomics approach to analyze the change in protein composition of the MT-associated protein (MAP) network by macrophage stimulation with the inflammatory cytokine interferon-γ and the endotoxin lipopolysaccharide. Overall the analysis identified 409 proteins that bound directly or indirectly to MTs. Of these, 52 were up-regulated 2-fold or greater and 42 were down-regulated 2-fold or greater after interferon-γ/lipopolysaccharide stimulation. Bioinformatics analysis based on publicly available binary protein interaction data produced a putative interaction network of MAPs in activated macrophages. We confirmed the up-regulation of several MAPs by immunoblotting and immunofluorescence analysis. More detailed analysis of one up-regulated protein revealed a role for HSP90β in stabilization of the MT cytoskeleton during macrophage activation.Microtubules (MTs)¹ are major structural components of the cytoskeleton that are intricately involved in cell morphology, motility, division, and intracellular organization and transport. The diverse roles of MTs are dependent on the polymer having the capacity to be both dynamic and static in nature. Individual MTs alternate between growing and shrinking by the rapid attachment and detachment of tubulin subunits at their ends (1, 2). Thus, MTs can continually reorganize and undergo cycles of growing, pausing, and shortening. A number of mechanisms exist to regulate this dynamic equilibrium and involve association of proteins with the MT lattice. MT-associated proteins (MAPs), such as MAP4 and tau, stabilize MTs by binding to the wall thus inhibiting MT disassembly (3, 4). Recently MT plus (+) end-binding proteins have been implicated in stabilizing MTs by associating with cortical proteins to tether the MT end to peripheral target sites (5–7). Stabilized MT subsets are biochemically distinct and acquire posttranslational modifications that can be used to differentiate them from dynamic subsets. For example, posttranslational modifications such as glutamylation (8), detyrosination (8, 9), and acetylation (10) occur on MTs that exhibit increased stability. Stabilized MTs have been implicated in MT transport by allowing increased binding of MT motors (11, 12). Numerous other MAPs have been shown to regulate MT form and function including control of MT nucleation and elongation, MT linkage to and movement of organelles, and modulation of MT growth to allow scaffolding of signal transduction events (13).The extensive MT network provides a large surface area to serve as a platform for the binding of a large number of proteins that is likely heavily influenced by local cellular events and cell type. Traditionally the term MAP referred to proteins that bind directly to tubulin within the MT polymer, and a lot of recent debate and controversy have surrounded the definition of a MAP (14, 15). In this and other reports the definition of MAPs is considered to also include proteins that indirectly or transiently interact with MTs, co-localize with MTs, or influence MT growth dynamics in some way (16). The advent of proteomics has allowed cytoskeleton researchers to resolve the spectrum of MAPs. To date, the MT proteome has been resolved by MS analysis in developmentally important animal and plant models including Xenopus laevis egg extracts (17), Drosophila melanogaster embryos (18), Artemia franciscana embryos (19), Arabidopsis suspension cells (20), and complex mammalian tissues such as rat brain (21). The MT proteome has also been described for specialized MT structures including mitotic spindles (22–24), centrosomes (25, 26), and cilia (27, 28).Macrophages are key regulators of the immune system connecting innate and specific immune responses. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, is a potent activator of monocytes and macrophages. LPS triggers the abundant secretion of many cytokines from macrophages including IL-1 (29), IL-6, (30), and tumor necrosis factor-α (31), which together contributes to the pathophysiology of septic shock. IFN-γ is a proinflammatory cytokine produced by the host in response to intracellular pathogens. IFN-γ binds to IFN-γ receptors on macrophages, and IFN-γ signaling induces the production and/or release of cytokines, like IL-1 or tumor necrosis factor-α, which enhance LPS-mediated effects (32). Thus, the synergy between LPS and inflammatory cytokines such as IFN-γ represents an important regulatory mechanism by which the host tackles a significant, ongoing infection before it activates potent effector responses (33). It has been demonstrated that LPS may cause changes in monocyte cytoskeleton and directly influence assembly of isolated MTs (34). Recently we observed that classical activation of murine resident peritoneal or RAW264.7 macrophages with a combination of IFN-γ and LPS induces an increase in stabilized cytoplasmic MTs (5). A significant effort has been made to unravel the importance of stable MTs in cellular processes over the past few years. With respect to macrophage function, stable MTs could potentially function as tracks for vesicle secretion of cytokines and matrix metalloproteinases necessary to effect the enhanced inflammatory response observed in classically activated macrophages. We recently demonstrated that stable MTs are important for cell spreading as well as the binding of large particles in activated macrophages (5). The stabilization of macrophage interphase MTs is uniquely rapid, thus serving as an ideal model for studying MAPs involved in MT modulation in mammalian cells.The focus of the present study was to identify the MT-associated proteins involved in altering and stabilizing MT structures and also to resolve the spectrum of proteins within the MT proteome of a mammalian cell. To achieve this goal, we used a proteomics approach involving a MAP purification technique based on MT co-sedimentation (35) followed by off-line fractionation and identification of MAPs using LC-MS/MS. Information provided by mass spectrometry analysis allowed us to analyze the changes in MAP abundance during activation of macrophages by IFN-γ/LPS. These studies also provided candidate proteins for selective molecular intervention for chronic inflammatory disorders.     

Reduced Uptake as a Mechanism of Zinc Tolerance in Oscillatoria anguistissima

Oscillatoria anguistissima could tolerate 50 ppm ZnSO₄.7H₂O, and a zinc-tolerant strain with maximum tolerance concentration (MTC) of 100 ppm ZnSO₄.7H₂O was obtained by stepwise transfer to higher concentrations. The adaptation was irreversible even after three generations in metal-free medium. In the presence of metal, the tolerant strain grew with a shorter lag period of 4 days as against 6 days in the case of the wild strain. The tolerant strain had higher MTC than that of the wild strain for other metals also, viz., Ni²⁺, Co²⁺, Cu²⁺ and Cd²⁺. The zinc resistance in the tolerant strain was a result of reduced uptake, since around 42% of the total metal was present on the surface as against only 30% in the wild strain. The calcium-stimulated uptake, as observed in the wild strain, was absent in the tolerant strain. Ultrastructural comparisons revealed no structural change in the tolerant strain on exposure to zinc, whereas in the wild strain a thick extracellular matrix was observed. Received: 25 January 2001 / Accepted: 9 March 2001     

Effect of cadmium uptake and heat stress on root ultrastructure, membrane damage and antioxidative response in rice seedlings

Excess cadmium (Cd²⁺) in the soil environment is taken up by plants and can cause phytotoxicity. Elevated temperatures also lead to deleterious effects on plants. Plants are very often exposed to a combination of stresses rather than a single stress. The effect of Cd²⁺ and heat stress (HS) on the growth, root ultrastructure, lipid peroxidation (MDA), hydrogen peroxide accumulation and the activities of antioxidant enzymes peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) of rice roots from sensitive cv. DR-92 and tolerant cv. Bh-1 were investigated at 10 and 20 day of growth under controlled conditions. At day 10 under all Cd²⁺ treatments, the Cd²⁺ content between the two rice cultivars were almost similar. Application of 500 μM Cd²⁺ significantly increased metal concentrations at day 20 in the roots of rice seedlings resulting in a maximum accumulation of 44.25 μg Cd²⁺ g^-1 dry wt in cv. DR-92 and 30 μg Cd²⁺ g^-1 dry wt in cv. Bh-1 with a ~25 % decline in Relative Growth Index (RGI) in cv. DR-92. TEM studies revealed slight disorganization with cell wall ingrowths in root tissues from cv. DR-92 grown in 100 μM Cd²⁺ + HS. Uptake and accumulation of Cd²⁺ increased upon heat treatment in parenchyma, vacuoles and vascular cylinder of root tissues. Peroxidase primarily located in cell walls, the intensity being higher in sensitive cv. DR-92. Under Cd²⁺ stress alone, plants of sensitive cv. DR-92 significantly increased the H₂O₂ and MDA levels together with increased activities of the enzymes POD, CAT and APX at day 10 but remained almost stable at day 20. A strong increase in MDA levels was noted at day 20 in tolerant cv. Bh-1. Cd²⁺ + HS treatments in tolerant cv.Bh-1 led to a decreased H₂O₂ and MDA levels and decreased activities of the enzymes POD, CAT and APX. Results suggest stimulation of root antioxidant system under combination of two stresses and that heat stress seem to have a direct protective role by mitigating the effect of mild Cd²⁺ toxicity largely by enhanced Cd²⁺-MT formation contributing thereby towards the management of Cd²⁺ toxicity at cellular level that confers Cd²⁺ tolerance to rice cv. Bh-1.     

Exploration of antimicrobial and antioxidant potential of newly synthesized 2,3-disubstituted quinazoline-4(3H)-ones

A series of 2-(chloromethyl)-3-(4-methyl-6-oxo-5-[(E)-phenyldiazenyl]-2-thioxo-5,6-dihydropyrimidine-1(2H)-yl)quinazoline-4(3H)-ones 9a-j was synthesized by treating 2-(chloroacetyl)amino benzoic acid with 3-amino-6-methyl-5-[(E)-phenyldiazenyl]-2-thioxo-2,5-dihydropyrimidine-4(3H)-one 8a-j and was screened for in vitro antibacterial activities against a representative panel of Gram-positive and Gram-negative bacteria. The compounds were synthesized in excellent yields and the structures were corroborated on the basis of IR, ¹H NMR, Mass and elemental analysis data. All the synthesized compounds elicited the potent inhibitory action against all the tested bacterial stains. Furthermore, in order to explore the antioxidant potential of newly synthesized compounds, the free radical scavenging activity measurement were performed by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay method. It is revealed from the antioxidant screening results that the compounds 9c and f manifested profound antioxidant potential.     

Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure

An early event in the pathogenesis of emphysema is the development of inflammation associated with accumulation of polymorphonuclear leukocytes (PMN) in small airways, and inflammatory cell recruitment from the circulation involves migration across endothelial and epithelial cell barriers. Platelet-activating factor (PAF) promotes transendothelial migration in several vascular beds, and we postulated that increased PAF production in the airways of smokers might enhance inflammatory cell recruitment and exacerbate inflammation. To examine this possibility, we incubated human lung microvascular endothelial cells (HMVEC-L) with cigarette smoke extract (CSE) and found that CSE inhibits PAF-acetylhydrolase (PAF-AH) activity. This enhances HMVEC-L PAF production and PMN adherence, and adherence is blocked by PAF receptor antagonists (CV3988 or ginkgolide B). CSE also inhibited PAF-AH activity of lung endothelial cells isolated from wild-type (WT) and iPLA(2)β knockout mice, and with WT cells, CSE enhanced PAF production and RAW 264.7 cell adherence. In contrast, CSE did not affect PAF production or RAW 264.7 cell adherence to iPLA(2)β-null cells, suggesting that iPLA(2)β plays an important role in PAF production by lung endothelial cells. These findings suggest that inhibition of PAF-AH by components of cigarette smoke may initiate or exacerbate inflammatory lung disease by enhancing PAF production and promoting accumulation of inflammatory cells in small airways. In addition, iPLA(2)β is identified as a potential target for therapeutic interventions to reduce airway inflammation and the progression of chronic lung disease.