TLR Signaling Paralyzes Monocyte Chemotaxis through Synergized Effects of p38 MAPK and Global Rap-1 Activation

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ∼0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP3 (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase

Synthesis, spectral LFER and antimicrobial activities of some (E)-N׳-(1-(substituted phenyl)ethylidene)benzohydrazides

About eleven substituted (E)-N׳-(1-(substituted phenyl) ethylidene) benzo- hydrazides have been synthesized. They are characterized by their analytical, ultraviolet, infrared and NMR spectral data. The antibacterial and fungal activities of these chalcones have been evaluated

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3

Young onset diabetes in Asian Indians is associated with lower measured and genetically determined beta-cell function:an INSPIRED study

Aims/hypothesis: South Asians in general, and Asian Indians in particular, have higher risk of type 2 diabetes compared with white Europeans, and a younger age of onset. The reasons for the younger age of onset in relation to obesity, beta cell function and insulin sensitivity are under-explored. Methods: Two cohorts of Asian Indians, the ICMR-INDIAB cohort (Indian Council of Medical Research-India Diabetes Study) and the DMDSC cohort (Dr Mohan’s Diabetes Specialties Centre), and one of white Europeans, the ESDC (East Scotland Diabetes Cohort), were used. Using a cross-sectional design, we examined the comparative prevalence of healthy, overweight and obese participants with young-onset diabetes, classified according to their BMI. We explored the role of clinically measured beta cell function in diabetes onset in Asian Indians. Finally, the comparative distribution of a partitioned polygenic score (pPS) for risk of diabetes due to poor beta cell function was examined. Replication of the genetic findings was sought using data from the UK Biobank. Results: The prevalence of young-onset diabetes with normal BMI was 9.3% amongst white Europeans and 24–39% amongst Asian Indians. In Asian Indians with young-onset diabetes, after adjustment for family history of type 2 diabetes, sex, insulin sensitivity and HDL-cholesterol, stimulated C-peptide was 492 pmol/ml (IQR 353–616, p&lt;0.0001) lower in lean compared with obese individuals. Asian Indians in our study, and South Asians from the UK Biobank, had a higher number of risk alleles than white Europeans. After weighting the pPS for beta cell function, Asian Indians have lower genetically determined beta cell function than white Europeans (p&lt;0.0001). The pPS was associated with age of diagnosis in Asian Indians but not in white Europeans. The pPS explained 2% of the variation in clinically measured beta cell function, and 1.2%, 0.97%, and 0.36% of variance in age of diabetes amongst Asian Indians with normal BMI, or classified as overweight and obese BMI, respectively. Conclusions/interpretation: The prevalence of lean BMI in young-onset diabetes is over two times higher in Asian Indians compared with white Europeans. This phenotype of lean, young-onset diabetes appears driven in part by lower beta cell function. We demonstrate that Asian Indians with diabetes also have lower genetically determined beta cell function

Cytisus scoparius link - A natural antioxidant

BACKGROUND: Recent investigations have shown that the antioxidant properties of plants could be correlated with oxidative stress defense and different human diseases. In this respect flavonoids and other polyphenolic compounds have gained the greatest attention. The plant Cytisus scoparius contains the main constituent of flavone and flavonals. The present study was undertaken to evaluate the in vitro antioxidant activities of extract of aerial part of Cytisus scoparius. METHODS: The plant extract was tested for DPPH (1, 1-diphenyl, 2-picryl hydrazyl) radical scavenging, nitric oxide radical scavenging, superoxide anion radical scavenging, hydroxyl radical scavenging, antilipid peroxidation assay, reducing power and total phenol content. RESULTS: The extract exhibited scavenging potential with IC(50 )value of 1.5 μg/ml, 116.0 μg/ml and 4.7 μg/ml for DPPH, nitric oxide and superoxide anion radicals. The values were found to lesser than those of vitamin C, rutin, and curcumin, as standards. The extract showed 50% protection at the dose of 104.0 μg/ml in lipid peroxidation induced by Fe(2+)/ ascorbate system in rat liver microsomal preparation. There is decrease in hydroxyl radical generation with IC(50 )value of 27.0 μg/ml when compared with standard vitamin E. The reducing power of the extract depends on the amount of extract. A significant amount of polyphenols could be detected by the equivalent to 0.0589 μg of pyrocatechol from 1 mg of extract. CONCLUSION: The results obtained in the present study indicate that hydro alcoholic extract of aerial part of Cytisus scoparius is a potential source of natural antioxidants

LPS treated human monocytes displayed flattened morphology and are inhibited for chemotaxis.

<p><b>A</b>) Relative chemotactic inhibition by different TLR ligands. Primary human monocytes were treated with the indicated TLR ligands exactly as described under <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030404#s4" target="_blank">Methods</a></b> and allowed to migrate towards 20 nM CCL2 or CXCL12 in the Trans-well chamber. Data are plotted as histograms with error bars (n = 3; **, p<0.03; ***, p<0.01). <b>B</b>) LPS dose response of chemotactic inhibition of monocytes. Monocytes (5×10⁵) were pretreated for 15 min with the various concentrations of LPS, washed 3 X and placed in the upper wells of 5.0 µm Trans-well and allowed to migrate towards 20 nM CCL2 or CXCL12 in the bottom wells. Relative fraction (%) of input cells that migrated to the bottom well is plotted in the histogram with error bar (n = 3; *, p<0.03, **, p<0.04). <b>C</b>) Chemotaxis of monocytes towards CCL2, CXCL12 or fMLF after preincubation with TLR2, TLR4 ligands in the presence of TLR2, TLR4 antibodies or isotype IgG control. Relative fraction (%) of migrated cells is plotted in the histogram with error bars. <b>D</b>) LPS pretreatment abolished chemokine induced F-actin polarizarion. Fresh monocytes on cover slips were treated with or without LPS (2 ng/ml) for 15 min at 37°C prior to 2 min stimulation with 20 nM CCL2 or CXCL12. Cells were fixed and stained with Alexa-488 conjugated phalloidin and examined by fluorescent microscopy and interference optics (DIC). <b>E</b>) Time course of morphological change in monocytes induced by various TLR ligands (LPS, 2 ng/ml; MALP-2, 200 ng/ml; and Pam₃CSK₄, 50 ng/ml). Cells were fixed and stained for F-actin as above. <b>F</b>) F-actin polarization was preserved in monocytes stimulated with 20 nM CCL2 prior to (top row) or simultaneously (bottom row) with LPS at 2 ng/ml. The middle row shows results with cells pretreated with LPS for 3 min before CCL2. Figures in <b>D</b>, <b>E</b> and <b>F</b> represent three independent experiments using monocytes from different donors.</p

LPS induced phosphorylation of MAPKs.

<p>p44/42 ERK (<b>A1–A3</b>) and p38 MAPK (<b>B1–B3</b>) were phosphorylated after CXCL12 (20 nM) stimulation or LPS (2 ng/ml) treatment. Human monocytes (2×10⁶ cells for each time point) treated with or without CXCL12 were collected at 1.5, 2, 3 and 5 min. Monocytes (5×10⁶ for each time point) treated with or without LPS were collected 0, 15, 30, 60 and 100 min. Half of the cells treated with or without LPS and all the cells treated with or without CXCL12 were extracted with RIPA buffer and proteins were resolved by 4–20% gradient SDS-PAGE. Phospho-ERK and total ERKs were detected by immunoblotting with phospho-ERK specific mAb, E10 and rabbit antibody against total ERK (<b>A1</b> and <b>A2</b>). Reacting with anti phospho-p38 MAPK (T180/Y182) mAb and rabbit antibody against total p38 detected phospho-p38 and total p38 respectively (<b>B1</b> and <b>B2</b>). Immunoblots are representative of results with monocytes from 3 donors. The remaining half of the cells treated with or without were stained with a mixture of Alexa-647 E10 mAb against phospho ERK (T202/Y204) and Alexa-488 28B10 mAb against phospho-p38 MAPK (T180/Y182) and analyzed by flow cytometry. Ratios of MFVs for the respective phosho-MAPKs in LPS treated vs. untreated cells are plotted as histograms (n = 5, * p<0.03, ** p<0.01). The remaining cells were extracted with RIPA buffer and proteins resolved by 4–20% gradient SDS-PAGE. Phospho-ERK and total ERKs were detected by immunoblotting with phospho-ERK specific mAb, E10 and rabbit antibody against total ERK. Reacting with anti phospho-p38 MAPK (T180/Y182) mAb and rabbit antibody against total p38 detected phospho-p38 and total p38 respectively (A3 and B3) Immunoblots are representative of results with monocytes from 5 donors.</p